Proteolytic Markers as Diagnostic Biomarkers for Cancer, Organ Injury and Muscle Rehabilitation/Exercise Overtraining

ABSTRACT

The present invention identifies biomarkers that are diagnostic of nerve cell injury, organ injury, and/or neuronal disorders. Detection of different biomarkers of the invention are also diagnostic of the degree of severity of nerve injury, the cell(s) involved in the injury, and the subcellular localization of the injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 11/106,932,filed Apr. 15, 2005, which claims the priority of U.S. ProvisionalPatent application No. 60/562,819 filed Apr. 15, 2004; which areincorporated herein by reference in their entirety.

The subject invention was made with government support under a researchproject supported by National Institutes of Health Grant #R01 NS39091,National Institutes of Health Grant #R01 NS40182, U.S. Army Grant #DAMD17-99-1-9565, and U.S. Army Grant #DAMD 17-01-1-0765.

FIELD OF THE INVENTION

The invention provides for the reliable detection and identification ofbiomarkers that are uniquely produced in brain injury and/or stress,neuronal disorders, organ injury and/or stress, cancer and cancertreatment, and muscle rehabilitation/exercise overtraining are importantfor the diagnosis and prognosis and the monitoring of damage of the sameorgan/tissue system of interest. The profile of proteolytic products orfragments of organ-enriched or specific proteins/peptides in patientswith damage to organ or tissue and cells within them are distinguishedfrom normal controls using inexpensive techniques. These techniquesprovide simple yet sensitive approaches to diagnosing damage or stressto the central and peripheral nervous system, to other organs or tomultiple organs, various cancers, cancer treatment, musclerehabilitation/exercise overtraining and other human pathological orstressed conditions where major organ(s) is compromised or altered usingbiological fluids.

BACKGROUND OF THE INVENTION

The incidence of traumatic brain injury (TBI) in the United States isconservatively estimated to be more than 2 million persons annually withapproximately 500,000 hospitalizations. Of these, about 70,000 to 90,000head injury survivors are permanently disabled. The annual economic costto society for care of head-injured patients is estimated at $25billion. These figures are for the civilian population only and theincidence is much greater when combat casualties are included. In modernwarfare (1993-2000), TBI is the leading cause of death (53%) amongwounded who have reached medical care facilities.

Assessment of pathology and neurological impairment immediately afterTBI is crucial for determination of appropriate clinical management andfor predicting long-term outcome. The outcome measures most often usedin head injuries are the Glasgow Coma Scale (GCS), the Glasgow OutcomeScale (GOS), computed tomography, and magnetic resonance imaging (MRI)to detect intracranial pathology. However, despite dramatically improvedemergency triage systems based on these outcome measures, most TBIsuffer long term impairment and a large number of TBI survivors areseverely affected despite predictions of “good recovery” on the GOS. Inaddition, CT and MRI are expensive and cannot be rapidly employed in anemergency room environment. Moreover, in austere medical environmentsassociated with combat, accurate diagnosis of TBI would be an essentialprerequisite for appropriate triage of casualties.

Accordingly, the neural pathways of a mammal are particularly at risk ifneurons are subjected to mechanical or chemical trauma or to neuropathicdegeneration sufficient to put the neurons that define the pathway atrisk of dying. A host of neuropathies, some of which affect only asubpopulation or a system of neurons in the peripheral or centralnervous systems have been identified to date. The neuropathies, whichmay affect the neurons themselves or the associated glial cells, mayresult from cellular metabolic dysfunction, infection, exposure to toxicagents, autoimmunity dysfunction, malnutrition or ischemia. In somecases the cellular dysfunction is thought to induce cell death directly.In other cases, the neuropathy may induce sufficient tissue necrosis tostimulate the body's immune/inflammatory system and the mechanisms ofthe body's immune response to the initial neural injury then destroysthe neurons and the pathway defined by these neurons.

Another common injury to the CNS is stroke, the destruction of braintissue as a result of intracerebral hemorrhage or infarction. Stroke isa leading cause of death in the developed world. It may be caused byreduced blood flow or ischemia that results in deficient blood supplyand death of tissues in one area of the brain (infarction). Causes ofischemic strokes include blood clots that form in the blood vessels inthe brain (thrombus) and blood clots or pieces of atherosclerotic plaqueor other material that travel to the brain from another location(emboli). Bleeding (hemorrhage) within the brain may also cause symptomsthat mimic stroke. The ability to detect such injury is lacking in theprior art.

Mammalian neural pathways also are at risk due to damage caused byneoplastic lesions. Neoplasias of both the neurons and glial cells havebeen identified. Transformed cells of neural origin generally lose theirability to behave as normal differentiated cells and can destroy neuralpathways by loss of function. In addition, the proliferating tumors mayinduce lesions by distorting normal nerve tissue structure, inhibitingpathways by compressing nerves, inhibiting cerebrospinal fluid or bloodsupply flow, and/or by stimulating the body's immune response.Metastatic tumors, which are a significant cause of neoplastic lesionsin the brain and spinal cord, also similarly may damage neural pathwaysand induce neuronal cell death.

Identification and development of proteolytic products as biomarkersand/or diagnostic markers has been primarily focused within the confinesof the CNS. Damage to peripheral nerves, such as occurred with diabeticneuropathies or administration of chemo-therapeutic agents such as thoseused to treat cancer, can also involve proteolytic damage similar tothat seen in the CNS. Moreover, since protease activation is a majortheme during cell injury in other organ injury or (such as liver,kidney, lung, gut, heart etc.), skeletal muscle overtraining and cancercell proliferation or chemotherapy-induced tumor cell death and tumorshrinkage, this invention has a board-based application in diagnosticsand monitoring of various organ injuries, multiple organ injury, CABG,sepsis, hyperventilation induced lung injury, muscle overtraining,cancer and the like.

There is thus, a need in the art appropriate, specific, inexpensive andsimple diagnostic clinical assessments of individual or multiple organinjury or stress, various organ-specific cancers and muscle training orovertraining, their severity and therapeutic treatment monitoring andefficacy assessment. Thus identification of proteolytic products ofproteins or peptides that are specific to or predominantly found in aspecific organ would prove immensely beneficial for both prediction ofoutcome and for guidance of targeted therapeutic delivery or muscletraining and rehabilitation monitoring.

SUMMARY

The invention provides for the reliable detection and identification ofbiomarkers that are uniquely produced in brain injury and/or stress,neuronal disorders, organ injury and/or stress, cancer and cancertreatment, and muscle rehabilitation/exercise overtraining are importantfor the diagnosis and prognosis and the monitoring of damage of the sameorgan/tissue system of interest. The profile of proteolytic products orfragments of organ-enriched or specific proteins/peptides in patientswith damage to organ or tissue and cells within them are distinguishedfrom normal controls using inexpensive techniques. These techniquesprovide simple yet sensitive approaches to diagnosing damage or stressto the nervous system, to other organs or to multiple organs, variouscancers, cancer treatment, muscle rehabilitation/exercise over-trainingand other human pathological or stressed conditions where major organ(s)is compromised or altered using biological fluids.

In a preferred embodiment, the invention provides biomarkers that areindicative of traumatic brain injury, neuronal damage, neural disorders,brain damage, neural damage due to drug or alcohol addiction, diseasesassociated with the brain or nervous system, such as the central andperipheral nervous systems (CNS, PNS). Preferably, the biomarkers areproteolytic enzymes which are activated as a result of damage to organssuch as for example: heart, brain, liver, kidneys, lung, gut; neurons,central nervous system, peripheral nervous system, as well as skeletalmuscles. Preferably the proteolytic enzymes are activated and cleavetarget proteins, peptides and fragments thereof due to neural and organinjury. Target proteins include, but are not limited to proteins,peptides or fragments thereof associated with neuronal cells, braincells or any cell that is present in the brain and central nervoussystems, organs such as heart, liver, kidneys and the like. Non-limitingexamples of proteolytic enzymes that are detected upon neural and/ororgan injury include (in alphabetical order): Achromopeptidase,Aminopeptidase, Ancrod, Angiotensin Converting Enzyme, Bromelain,Calpain, Calpain I, Calpain II, Carboxypeptidase A, Carboxypeptidase B,Carboxypeptidase G, Carboxypeptidase P, Carboxypeptidase W,Carboxypeptidase Y, Caspase, Caspase 1, Caspase 2, Caspase 3, Caspase 4,Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10,Caspase 13, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin G.Cathepsin H, Cathepsin L, Chymopapain, Chymase, Chymotrypsin,α-Clostripain, Collagenase, Complement C1r, Complement C1s, ComplementFactor D, Complement factor I, Cucumisin, Dipeptidyl peptidase IV,Elastase, leukocyte, Elastase, pancreatic, Endoproteinase Arg-C,Endoproteinase Asp-N, Endoproteinase Glu-C, Endoproteinase Lys-C,Enterokinase, Factor Xa, Ficin, Furin, Granzyme A, Granzyme B, HIVProtease, Igase, Kallikrein tissue, Kinase, Leucine Aminopeptidase(General), Leucine aminopeptidase, cytosol, Leucine aminopeptidase,microsomal, Matrix metalloprotease, Methionine Aminopeptidase, Neutrase,Papain, Pepsin, Plasmin, Prolidase, Pronase E, Prostate SpecificAntigen, Protease, Protease S, Proteasomes, Proteinase, Proteinase 3,Proteinase A, Proteinase K, Protein C, Pyroglutamate aminopeptidase,Renin, Rennin, Thrombin, Tissue Plasminogen Activator, Troponins,Trypsin, Tryptase, Urokinase. Preferably, any one of SEQ ID NO's.: 1-148are also detected.

In another preferred embodiment, the proteolytic enzyme biomarkers havea specific activity for the protein substrates, for example the nonlimiting examples listed in Table 1, of about 1 μg to about 500 μg per 1mg of substrate protein per being proteolyzed in injured or stressedorgans (in vivo) within minutes to days after or in vitro using purifiedprotease-substrate protein/protein mixture ratio of 1/10,000 to 1/20 ata time point within minutes to hours.

In a preferred embodiment the biomarkers are activated upon injury of,for example an organ, neuronal cells, and result in the proteolysis ofproteins, peptides associated with the organ, neuronal cells. Examplesof preferred proteins include but not limited to: troponins such ascardiac or muscle troponins, such as, for example, troponin I,troponin-T, troponin-C; neural peptides, include, but are not limited topeptides of axonal proteins, amyloid precursor protein, dendriticproteins, somal proteins, presynaptic proteins, post-synaptic proteinsfragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders are identified by SEQ ID NO's:1-148.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS injury or neural disorders are about 50% homologous topeptides identified by SEQ ID NO's: 1-148, preferably the peptidesidentified as targets for proteolytic enzyme biomarkers for diagnosisand detection of brain and/or CNS injury or neural disorders are about70% homologous to peptides identified by SEQ ID NO's: 1-148, preferablythe peptides identified as targets for proteolytic enzyme biomarkers fordiagnosis and detection of brain and/or CNS injury or neural disordersare about 80% homologous to peptides identified by SEQ ID NO's: 1-148,preferably the peptides identified as targets for proteolytic enzymebiomarkers for diagnosis and detection of brain and/or CNS injury orneural disorders are about 90%, 95%, 96%, 97%, 95%, 99% or 99.9%homologous to peptides identified by SEQ ID NO's: 1-148.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS injury or neural disorders are at least about 10 amino acidslonger either at the N-Terminal and/or C-Terminal of the peptidesidentified by SEQ ID NO's: 1-148, preferably the peptides identified astargets for proteolytic enzyme biomarkers for diagnosis and detection ofbrain and/or CNS injury or neural disorders are at least about 20 aminoacids longer either at the N-Terminal and/or C-Terminal of the peptidesidentified by SEQ ID NO's: 1-148, preferably the peptides identified astargets for proteolytic enzyme biomarkers for diagnosis and detection ofbrain and/or CNS injury or neural disorders are at least about 50 aminoacids longer either at the N-Terminal and/or C-Terminal of the peptidesidentified by SEQ ID NO's: 1-148, preferably the peptides identified astargets for proteolytic enzyme biomarkers for diagnosis and detection ofbrain and/or CNS injury or neural disorders are at least about 80 aminoacids longer either at the N-Terminal and/or C-Terminal of the peptidesidentified by SEQ ID NO's: 1-148, preferably the peptides identified astargets for proteolytic enzyme biomarkers for diagnosis and detection ofbrain and/or CNS injury or neural disorders are at least about 100 aminoacids longer either at the N-Terminal and/or C-Terminal of the peptidesidentified by SEQ ID NO's: 1-148, preferably the peptides identified astargets for proteolytic enzyme biomarkers for diagnosis and detection ofbrain and/or CNS injury or neural disorders are at least about 200 aminoacids longer either at the N-Terminal and/or C-Terminal of the peptidesidentified by SEQ ID NO's: 1-148, preferably the peptides identified astargets for proteolytic enzyme biomarkers for diagnosis and detection ofbrain and/or CNS injury or neural disorders are up to about 400 aminoacids longer either at the N-Terminal and/or C-Terminal of the peptidesidentified by SEQ ID NO's: 1-148. Examples of longer amino acids arefound in Table 1, along with their accession numbers. The desired aminoacids to be included at either the N- or C-terminal of each biomarkeridentified by SEQ ID NO's.: 1-148 are thus, readily determined.

In a preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are, derived fromaxonal proteins such as for example, NF-200 (NF-H), NF-160 (NF-M), NF-68(NF-L), peptides, fragments or derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS injury or neural disorders, preferably are peptides ofamyloid precursor protein fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS injury or neural disorders, preferably are dendriticpeptides, such as for example: peptides of alpha-tubulin (P02551),beta-tubulin (P0 4691), MAP-2A/B, MAP-2C, Tau, Dynamin-1 (P21575),Dynactin (Q13561), P24.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are somalpeptides, for example: peptides, of UCH-L1 (Q00981), PEBP (P31044), NSE(P07323), Thy 1.1, Prion, Huntington fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are presynapticpeptides of synapsin-1, synapsin-2, alpha-synuclein (P37377),beta-synuclein (Q63754), GAP43, synaptophysin, synaptotagmin (P21707),syntaxin fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are post-synapticpeptides derived from PSD95, PSD93, NMDA-receptor (including allsubtypes).

In another preferred embodiment; peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are demyelinationpeptides, such as for example, peptides of myelin basic protein (MBP),myelin proteolipid protein, fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are glialpeptides, for example, peptides of GFAP (P47819), protein disulfideisomerase (PDI-P04785), fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are cholinergicpeptides, such as for example, peptides of acetylcholine esterase,choline acetyltransferase, fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are dopaminergicpeptides, such as for example, peptides of tyrosine hydroxylase (TH),phospho-TH, DARPP32, fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are noradrenergicpeptides, such as for example, peptides of dopamine beta-hydroxylase(DbH), fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are serotonergicpeptides, such as for example, peptides of tryptophan hydroxylase (TrH)fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are glutamatergicpeptides, such as for example, peptides of glutaminase, glutaminesynthetase, fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are, GABAergicpeptides, such as for example, peptides of GABA transaminase(4-aminobutyrate-2-ketoglutarate transaminase [GABAT]), glutamic aciddecarboxylase (GAD25, 44, 65, 67) fragments and derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are,neurotransmitter peptide receptors, such as for example, peptides ofbeta-adrenoreceptor subtypes, (e.g. beta (2)), alpha-adrenoreceptorsubtypes, (e.g. (alpha (2c)), GABA receptors (e.g. GABA(B)),metabotropic glutamate receptor (e.g. mGluR3), NMDA receptor subunits(e.g. NR1A2B), Glutamate receptor subunits (e.g. GluR4), 5-HT serotoninreceptors (e.g. 5-HT(3)), dopamine receptors (e.g. D4), muscarinic Achreceptors (e.g. M1), nicotinic acetylcholine receptor (e.g. alpha-7),fragments or derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, preferably are,neurotransmitter transporter peptides, such as for example, peptides ofnorepinephrine transporter (NET), dopamine transporter (DAT), serotonintransporter (SERT), vesicular transporter proteins (VMAT1 and VMAT2),GABA transporter vesicular inhibitory amino acid transporter(VIAAT/VGAT), glutamate transporter (e.g. GLT1), vesicular acetylcholinetransporter, choline transporter (e.g. CHT1), fragments, or derivativesthereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of brainand/or CNS/PNS injury or neural disorders, include, but are not limitedto vimentin (P31000), CK-BB (P07335), 14-3-3-epsilon (P42655), MMP2,MMP9, fragments or derivatives thereof.

In another preferred embodiment, peptides identified as targets forproteolytic enzyme biomarkers for diagnosis and detection of cardiacinjury are for example troponins, such as troponin-I, troponin-T andtroponin-C.

In another preferred embodiment, proteolytic enzyme biomarkers aredetected from samples of a patient who is susceptible to or sufferingfrom cancer, neuronal injury, and/or organ injury.

The markers are characterized by molecular weight, enzyme digestedfingerprints and by their known protein identities. The markers can beresolved from other peptides in a sample by using a variety offractionation techniques, e.g., chromatographic separation coupled withmass spectrometry, or by traditional immunoassays. In preferredembodiments, the method of resolution involves Surface-Enhanced LaserDesorption/Ionization (“SELDI”) mass spectrometry, in which the surfaceof the mass spectrometry probe comprises adsorbents that bind themarkers.

In another preferred embodiment, the presence of certain proteolyticenzyme biomarkers is indicative of the extent of CNS/PNS and/or braininjury. For example, detection of one or more dendritic damage markers,soma injury markers, demyelination markers, axonal injury markers wouldbe indicative of CNS injury and the presence of one or more would beindicative of the extent of nerve injury.

In another preferred embodiment, the presence of certain degraded neuralproteins is indicative of proteolytic enzyme activity and is indicativeof a neurological disorder, i.e. dendritic damage markers, soma injurymarkers, demyelination markers, axonal injury markers, synaptic terminalmarkers, post-synaptic markers.

Preferred methods for detection and diagnosis of CNS/PNS and/or braininjury comprise detecting at least one or more proteolytic enzymebiomarkers in a subject sample, and; correlating the detection of one ormore proteolytic enzyme biomarkers with a diagnosis of CNS and/or braininjury, wherein the correlation takes into account the detection of oneor more proteolytic enzyme biomarker in each diagnosis, as compared tonormal subjects. Preferably, the proteolytic enzyme biomarkers arespecific for example, neuronal proteins, tumor antigens, wherein the oneor more proteolytic enzyme biomarkers degrade proteins selected from:neural proteins, such as for example, axonal proteins—NF-200 (NF-H),NF-160 (NF-M), NF-68 (NF-L); amyloid precursor protein; dendriticproteins—alpha-tubulin (P02551), beta-tubulin (P04691), MAP-2A/B,MAP-2C, Tau, Dynamin-1 (P21575), Dynactin (Q13561), P24; somalproteins—UCH-L1 (Q00981), PEBP (P31044), NSE (P07323), Thy 1.1, Prion,Huntington; presynaptic proteins—synapsin-1, synapsin-2, alpha-synuclein(p37377), beta-synuclein (Q63754), GAP43, synaptophysin, synaptotagmin(P21707), syntaxin; post-synaptic proteins —PSD95, PSD93, NMDA-receptor(including all subtypes); demyelination biomarkers—myelin basic protein(MBP), myelin proteolipid protein; glial proteins—GFAP (P47819), proteindisulfide isomerase (PDI-P04785); neurotransmitterbiomarkers—cholinergic biomarkers: acetylcholine esterase, cholineacetyltransferase; dopaminergic biomarkers—tyrosine hydroxylase (TH),phospho-TH, DARPP32; noradrenergic biomarkers—dopamine beta-hydroxylase(DbH); serotonergic biomarkers—tryptophan hydroxylase (TrH);glutamatergic biomarkers—glutaminase, glutamine synthetase; GABAergicbiomarkers—GABA transaminase (4-aminobutyrate-2-ketoglutaratetransaminase [GABAT]), glutamic acid decarboxylase (GAD25, 44, 65, 67);neurotransmitter receptors—beta-adrenoreceptor subtypes, (e.g. beta(2)), alpha-adrenoreceptor subtypes, (e.g. (alpha (2c)), GABA receptors(e.g. GABA(B)), metabotropic glutamate receptor (e.g. mGluR3), NMDAreceptor subunits (e.g. NR1A2B), Glutamate receptor subunits (e.g.GluR4), 5-HT serotonin receptors (e.g. 5-HT(3)), dopamine receptors(e.g. D4), muscarinic Ach receptors (e.g. M1), nicotinic acetylcholinereceptor (e.g. alpha-7); neurotransmitter transporters—norepinephrinetransporter (NET), dopamine transporter (DAT), serotonin transporter(SERT), vesicular transporter proteins (VMAT1 and VMAT2), GABAtransporter vesicular inhibitory amino acid transporter (VIAAT/VGAT),glutamate transporter (e.g. GLT1), vesicular acetylcholine transporter,choline transporter (e.g. CHT1); other protein biomarkers include, butnot limited to vimentin (P31000), CK-BB (P07335), 14-3-3-epsilon(P42655), MMP2, MMP9.

In another preferred embodiment, antibodies specific for the proteolyticproducts of enzyme biomarkers bind to epitopes as identified by SEQ IDNO's: 1-148.

In another preferred embodiment, antibodies specific for the proteolyticproducts of enzyme biomarkers bind to epitopes that are about 50%homologous to sequences identified by SEQ ID NO's: 1-148, morepreferably antibodies specific for the proteolytic products of enzymebiomarkers bind to epitopes that are about 70% homologous to sequencesidentified by SEQ ID NO's: 1-148, more preferably antibodies specificfor the proteolytic products of enzyme biomarkers bind to epitopes thatare about 80% homologous to sequences identified by SEQ ID NO's: 1-148,more preferably antibodies specific for the proteolytic products ofenzyme biomarkers bind to epitopes that are about 90%, 95%, 96%, 97%,95%, 99% or 99.9% homologous to sequences identified by SEQ ID NO's:1-148.

In another preferred embodiment, antibodies specific for the proteolyticproducts of enzyme biomarkers bind to epitopes at least about 10 aminoacids longer at either the N-terminal and/or C-terminal of the epitopesas identified by SEQ ID NO's: 1-148, more preferably, antibodiesspecific for the proteolytic products of enzyme biomarkers bind toepitopes at least about 20 amino acids longer at either the N-terminaland/or C-terminal of the epitopes as identified by SEQ ID NO's: 1-148,more preferably, antibodies specific for the proteolytic products ofenzyme biomarkers bind to epitopes at least about 50 amino acids longerat either the N-terminal and/or C-terminal of the epitopes as identifiedby SEQ ID NO's: 1-148, more preferably, antibodies specific for theproteolytic products of enzyme biomarkers bind to epitopes at leastabout 100 amino acids longer at either the N-terminal and/or C-terminalof the epitopes as identified by SEQ ID NO's: 1-148, more preferably,antibodies specific for the proteolytic products of enzyme biomarkersbind to epitopes at least about 200 amino acids longer at either theN-terminal and/or C-terminal of the epitopes as identified by SEQ IDNO's: 1-148, more preferably, antibodies specific for the proteolyticproducts of enzyme biomarkers bind to epitopes up to at least about 500amino acids longer at either the N-terminal and/or C-terminal of theepitopes as identified by SEQ ID NO's: 1-148. Examples of longer aminoacids are found in Table 1, along with their accession numbers. Thedesired amino acids to be included at either the N- or C-terminal ofeach biomarker identified by SEQ ID NO's.: 1-148 are thus, readilydetermined and antibodies can be produced.

In another preferred embodiment, the invention provides a kit foranalyzing cell damage in a subject. The kit, preferably includes: (a) acomposition or panel of biomarkers as identified by anyone of SEQ IDNO's.: 1-148; (b) a substrate for holding a biological sample isolatedfrom a human subject suspected of having a damaged nerve cell, (c) anagent that specifically binds at least one or more of the proteolyticenzymes; and (d) printed instructions for reacting the agent with thebiological sample or a portion of the biological sample to detect thepresence or amount of at least one marker in the biological sample.

Preferably, the biological sample is a fluid in communication with thenervous system of the subject prior to being isolated from the subject;for example, CSF or blood, and the agent can be an antibody, aptamer, orother molecule that specifically binds at least one or more of theproteolytic enzymes. The kit can also include a detectable label such asone conjugated to the agent, or one conjugated to a substance thatspecifically binds to the agent (e.g., a secondary antibody).

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

FIG. 1 is a diagrammatic illustration showing an example of a methodrelating to diagnostics and therapy following organ stress or injury.Organ stress or injury and tumor formation or treatment induce increasedproteolytic activities which result in unique tissue protein fragmentsof diagnostic values.

FIGS. 2A-2E are gels showing examples of neural proteins subjected toproteolytic attack 48 h after traumatic brain injury in rat hippocampus.These proteins include NF-H (FIG. 2A), NF-L (FIG. 2B), Tau (FIG. 2C),APP (FIG. 2D) and βII-spectrin (FIG. 2E). Major breakdown products(BDPs) with their relative molecular weight are indicated. Ipsilateralcortical samples were also analyzed and they showed very similarpatterns of proteolysis.

FIGS. 3A and 3B are gels showing examples of Myelin proteins (MBP, FIG.3A; MOSP, FIG. 3B) being cleaved 24 hours after traumatic brain injury.

FIGS. 4A-4C are gels showing examples of production of polyclonal andmonoclonal that specifically detects an unique new N-terminal of atissue protein fragment (MBP-BDP). Naïve and TBI ipsilateral cortexsamples (48 hr after injury) were analyzed on immunoblots probed withanti-total MBP antibody (FIG. 4A), anti-MBP fragment-specific rabbitpolyclonal (FIG. 4B): or mouse monoclonal (FIG. 4C) antibodies. In FIG.4A, anti-total MBP antibody detects intact MBP-21 kDa and MBP-18 kDa innaïve brain and the 12 kDa and 10 kDa fragments (BDP-12 kDa, BDP-10 kDa)in the TBI brain. In FIGS. 4B and 4C, only BDP-12 kDa and BDP-10 kDawere detected, no intact MBP's (18 kDa and 21 kDa) were detected withthese antibodies, demonstrating their high selectivity for the in vivogenerated MBP fragments.

FIG. 5 shows gels of examples of Synaptic proteins (Synaptotagmin andSynaptojanin-1) being degraded in rat cortex and/or hippocampus 48 hrafter TBI.

FIG. 6 shows gels of the results obtained when human cardiac Troponin-T2and Troponin-I3 cleaved by calpain-2 and caspase-3 proteases, producingunique breakdown products (designated by a star).

FIG. 7 shows gels of the results obtained when human muscleTroponin-T1/3 was digested by calpain-1 protease, producing uniquebreakdown products. The Coomassie stained PVDF membrane was used toexcise bands for N-terminal sequencing. Western blot showsidentification of most of the bands as specific TnT BDPs. The map belowindicates the newly identified cleavage sites, one in TnT1 and one inTnT2.

DETAILED DESCRIPTION

The present invention identifies biomarkers that are diagnostic of CNS,muscle or other organ cell injury and/or stress and/or neuronaldisorders. Detection of different biomarkers of the invention are alsodiagnostic of the degree of severity of nerve injury, the cell(s)involved in the injury, and the subcellular localization of the injury.In particular, the invention employs a step of correlating the presenceor amount of one or more proteolytic enzymes which are activated by thepresence of peptide(s) from neural cells and/or organs due to injury.The presence of proteolytic enzymes is correlated with the severityand/or type of nerve cell injury and/or organ. The activity of aproteolytic enzyme and the generation of tissue protein breakdownproducts directly relate to severity of nerve tissue and/or organ injuryas a more severe injury damages a greater number of cells which in turncauses a larger amount of neural or cellular peptide(s) to accumulate inthe biological sample (e.g., CSF), thereby activating the proteolyticenzymes.

DEFINITIONS

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

“Marker” in the context of the present invention refers to a polypeptide(of a particular apparent molecular weight) which is differentiallypresent in a sample taken from patients having neural injury and/orneuronal disorders as compared to a comparable sample taken from controlsubjects (e.g., a person with a negative diagnosis, normal or healthysubject).

“Activity” of an enzyme is the amount of product produced per unit timeat a fixed temperature and pH.

“Specific activity” of an enzyme is the amount of product produced perunit time per mg protein.

“Substrate” is the target protein that the enzyme catalyzes. TheInternational Union of Biochemistry (I.U.B.) initiated standards ofenzyme nomenclature which recommend that enzyme names indicate both thesubstrate acted upon and the type of reaction catalyzed. For example,under this system, the enzyme uricase is called urate: O₂oxidoreductase, while the enzyme glutamic oxaloacetic transaminase (GOT)is called L-aspartate: 2-oxoglutarate aminotransferase.

“Complementary” in the context of the present invention refers todetection of at least two biomarkers, which when detected togetherprovides increased sensitivity and specificity as compared to detectionof one biomarker alone.

The phrase “differentially present” refers to differences in thequantity and/or the frequency of a marker present in a sample taken frompatients having for example, neural injury as compared to a controlsubject. For example, a marker can be a polypeptide which is present atan elevated level or at a decreased level in samples of patients withneural injury compared to samples of control subjects. Alternatively, amarker can be a polypeptide which is detected at a higher frequency orat a lower frequency in samples of patients compared to samples ofcontrol subjects. A marker can be differentially present in terms ofquantity, frequency or both.

A polypeptide is differentially present between the two samples if theamount of the polypeptide in one sample is statistically significantlydifferent from the amount of the polypeptide in the other sample. Forexample, a polypeptide is differentially present between the two samplesif it is present at least about 120%, at least about 130%, at leastabout 150%, at least about 180%, at least about 200%, at least about300%, at least about 500%, at least about 700%, at least about 900%, orat least about 1000% greater than it is present in the other sample, orif it is detectable in one sample and not detectable in the other.

Alternatively or additionally, a polypeptide is differentially presentbetween the two sets of samples if the frequency of detecting thepolypeptide in samples of patients' suffering from neural injury and/orneuronal disorders is statistically significantly higher or lower thanin the control samples. For example, a polypeptide is differentiallypresent between the two sets of samples if it is detected at least about120%, at least about 130%, at least about 150%, at least about 180%, atleast about 200%, at least about 300%, at least about 500%, at leastabout 700%, at least about 900%, or at least about 1000% more frequentlyor less frequently observed in one set of samples than the other set ofsamples.

“Diagnostic” means identifying the presence or nature of a pathologiccondition. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of “true positives”).Diseased individuals not detected by the assay are “false negatives.”Subjects who are not diseased and who test negative in the assay, aretermed “true negatives.” The “specificity” of a diagnostic assay is 1minus the false positive rate, where the “false positive” rate isdefined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

A “test amount” of a marker refers to an amount of a marker present in asample being tested. A test amount can be either in absolute amount(e.g., μg/ml) or a relative amount (e.g., relative intensity ofsignals).

A “diagnostic amount” of a marker refers to an amount of a marker in asubject's sample that is consistent with a diagnosis of neural injuryand/or neuronal disorder. A diagnostic amount can be either in absoluteamount (e.g., μg/ml) or a relative amount (e.g., relative intensity ofsignals).

A “control amount” of a marker can be any amount or a range of amountwhich is to be compared against a test amount of a marker. For example,a control amount of a marker can be the amount of a marker in a personwithout neural injury and/or neuronal disorder. A control amount can beeither in absolute amount (e.g., μg/ml) or a relative amount (e.g.,relative intensity of signals).

“Probe” refers to a device that is removably insertable into a gas phaseion spectrometer and comprises a substrate having a surface forpresenting a marker for detection. A probe can comprise a singlesubstrate or a plurality of substrates.

“Substrate” or “probe substrate” refers to a solid phase onto which anadsorbent can be provided (e.g., by attachment, deposition, etc.).

“Adsorbent” refers to any material capable of adsorbing a marker. Theterm “adsorbent” is used herein to refer both to a single material(“monoplex adsorbent”) (e.g., a compound or functional group) to whichthe marker is exposed, and to a plurality of different materials(“multiplex adsorbent”) to which the marker is exposed. The adsorbentmaterials in a multiplex adsorbent are referred to as “adsorbentspecies.” For example, an addressable location on a probe substrate cancomprise a multiplex adsorbent characterized by many different adsorbentspecies (e.g., anion exchange materials, metal chelators, orantibodies), having different binding characteristics. Substratematerial itself can also contribute to adsorbing a marker and may beconsidered part of an “adsorbent.”

“Adsorption” or “retention” refers to the detectable binding between anabsorbent and a marker either before or after washing with an eluant(selectivity threshold modifier) or a washing solution.

“Eluant” or “washing solution” refers to an agent that can be used tomediate adsorption of a marker to an adsorbent. Eluants and washingsolutions are also referred to as “selectivity threshold modifiers.”Eluants and washing solutions can be used to wash and remove unboundmaterials from the probe substrate surface.

“Resolve,” “resolution,” or “resolution of marker” refers to thedetection of at least one marker in a sample. Resolution includes thedetection of a plurality of markers in a sample by separation andsubsequent differential detection. Resolution does not require thecomplete separation of one or more markers from all other biomoleculesin a mixture. Rather, any separation that allows the distinction betweenat least one marker and other biomolecules suffices.

“Gas phase ion spectrometer” refers to an apparatus that measures aparameter which can be translated into mass-to-charge ratios of ionsformed when a sample is volatilized and ionized. Generally ions ofinterest bear a single charge, and mass-to-charge ratios are oftensimply referred to as mass. Gas phase ion spectrometers include, forexample, mass spectrometers, ion mobility spectrometers, and total ioncurrent measuring devices.

“Mass spectrometer” refers to a gas phase ion spectrometer that includesan inlet system, an ionization source, an ion optic assembly, a massanalyzer, and a detector.

“Laser desorption mass spectrometer” refers to a mass spectrometer whichuses laser as means to desorb, volatilize, and ionize an analyte.

“Detect” refers to identifying the presence, absence or amount of theobject to be detected.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, ³⁵S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavidin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogenic, orfluorescent signal, that can be used to quantify the amount of bounddetectable moiety in a sample. Quantitation of the signal is achievedby, e.g., scintillation counting, densitometry, or flow cytometry.

“Antibody” refers to a polypeptide ligand substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an epitope (e.g., an antigen). Therecognized immunoglobulin genes include the kappa and lambda light chainconstant region genes, the alpha, gamma, delta, epsilon and mu heavychain constant region genes, and the myriad immunoglobulin variableregion genes. Antibodies exist, e.g., as intact immunoglobulins or as anumber of well characterized fragments produced by digestion withvarious peptidases. This includes, e.g., Fab′ and F(ab)′₂ fragments. Theterm “antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It also includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies, humanizedantibodies, or single chain antibodies. “Fe” portion of an antibodyrefers to that portion of an immunoglobulin heavy chain that comprisesone or more heavy chain constant region domains, CH₁, CH₂ and CH₃, butdoes not include the heavy chain variable region.

“Immunoassay” is an assay that uses an antibody to specifically bind anantigen (e.g., a marker). The immunoassay is characterized by the use ofspecific binding properties of a particular antibody to isolate, target,and/or quantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to marker NF-200 from specific species such as rat, mouse, orhuman can be selected to obtain only those polyclonal antibodies thatare specifically immunoreactive with marker NF-200 and not with otherproteins, except for polymorphic variants and alleles of marker NF-200.This selection may be achieved by subtracting out antibodies thatcross-react with marker NF-200 molecules from other species. A varietyof immunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988), for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

“Energy absorbing molecule” or “EAM” refers to a molecule that absorbsenergy from an ionization source in a mass spectrometer thereby aidingdesorption of analyte, such as a marker, from a probe surface. Dependingon the size and nature of the analyte, the energy absorbing molecule canbe optionally used. Energy absorbing molecules used in MALDI arefrequently referred to as “matrix.” Cinnamic acid derivatives, sinapinicacid (“SPA”), cyano hydroxy cinnamic acid (“CHCA”) and dihydroxybenzoicacid are frequently used as energy absorbing molecules in laserdesorption of bioorganic molecules.

“Sample” is used herein in its broadest sense. A sample comprisingpolynucleotides, polypeptides, peptides, antibodies fragments andderivatives thereof, may comprise a bodily fluid; a soluble fraction ofa cell preparation, or media in which cells were grown; a chromosome, anorganelle, or membrane isolated or extracted from a cell; genomic DNA,RNA, or cDNA, polypeptides, or peptides in solution or bound to asubstrate; a cell; a tissue; a tissue print; a fingerprint, skin orhair; fragments and derivatives thereof.

“Substantially purified” refers to nucleic acid molecules or proteinsthat are removed from their natural environment and are isolated orseparated, and are at least about 60% free, preferably about 75% free,and most preferably about 90% free, from other components with whichthey are naturally associated.

“Substrate” refers to any rigid or semi-rigid support to which nucleicacid molecules or proteins are bound and includes membranes, filters,chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels,capillaries or other tubing, plates, polymers, and microparticles with avariety of surface forms including wells, trenches, pins, channels andpores.

As used herein, the term “fragment or segment”, as applied to a nucleicacid sequence, gene or polypeptide, will ordinarily be at least about 5contiguous nucleic acid bases (for nucleic acid sequence or gene) oramino acids (for polypeptides), typically at least about 10 contiguousnucleic acid bases or amino acids, more typically at least about 20contiguous nucleic acid bases or amino acids, usually at least about 30contiguous nucleic acid bases or amino acids, preferably at least about40 contiguous nucleic acid bases or amino acids, more preferably atleast about 50 contiguous nucleic acid bases or amino acids, and evenmore preferably at least about 60 to 80 or more contiguous nucleic acidbases or amino acids in length. “Overlapping fragments” as used herein,refer to contiguous nucleic acid or peptide fragments which begin at theamino terminal end of a nucleic acid or protein and end at the carboxyterminal end of the nucleic acid or protein. Each nucleic acid orpeptide fragment has at least about one contiguous nucleic acid or aminoacid position in common with the next nucleic acid or peptide fragment,more preferably at least about three contiguous nucleic acid bases oramino acid positions in common, most preferably at least about tencontiguous nucleic acid bases amino acid positions in common.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least 20nucleotides, more generally at least 23 nucleotides, ordinarily at least26 nucleotides, more ordinarily at least 29 nucleotides, often at least32 nucleotides, more often at least 35 nucleotides, typically at least38 nucleotides, more typically at least 41 nucleotides, usually at least44 nucleotides, more usually at least 47 nucleotides, preferably atleast 50 nucleotides, more preferably at least 53 nucleotides, and inparticularly preferred embodiments will be at least 56 or morenucleotides.

As used herein, the terms “polypeptide” or “peptide” encompasses aminoacid chains of any length, including full length proteins recitedherein.

As used herein, “peptides or epitopes with longer amino sequences”encompasses amino acid chains of any length, including full lengthproteins recited herein. Preferably, the antibodies produced bindepitopes that comprise at least about 3 amino acids long. In otherpreferred embodiments, the term “the proteolytic products of enzymebiomarkers bind to epitopes at least about 10 amino acids longer thanthe epitopes as identified by SEQ ID NO's: 1-148” includes an amino acidchain of 10 amino acids at the amino-terminal and/or the carboxyterminal of a desired peptide. Examples of longer amino acids are foundin Table 1, along with their accession numbers. The desired amino acidsto be included at either the N- or C-terminal of each biomarkeridentified by SEQ ID NO's.: 1-148 are thus, readily determined.

As used herein, “variant” or “derivative” of polypeptides refers to anamino acid sequence that is altered by one or more amino acid residues.The variant may have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties (e.g., replacement ofleucine with isoleucine). More rarely, a variant may have“nonconservative” changes (e.g., replacement of glycine withtryptophan). Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, LASERGENE software (DNASTAR).

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base.

“Stringency” is meant the combination of conditions to which nucleicacids are subject that cause the duplex to dissociate, such astemperature, ionic strength, and concentration of additives such asformamide. Conditions that are more likely to cause the duplex todissociate are called “higher stringency”, e.g. higher temperature,lower ionic strength and higher concentration of formamide.

For applications requiring high selectivity, one will typically desireto employ relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.10 M NaCl at temperatures ofabout 50° C. to about 70° C.

For certain applications, it is appreciated that lower stringencyconditions are required. Under these conditions, hybridization may occureven though the sequences of probe and target strand are not perfectlycomplementary, but are mismatched at one or more positions. Conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. For example, a medium stringency condition couldbe provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C.to about 55° C., while a low stringency condition could be provided byabout 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. Thus, hybridization conditions can be readilymanipulated depending on the desired results.

The phrase “hybridizing conditions” and its grammatical equivalents,when used with a maintenance time period, indicates subjecting thehybridization reaction admixture, in context of the concentration of thereactants and accompanying reagents in the admixture, to time,temperature, pH conditions sufficient to allow the polynucleotide probeto anneal with the target sequence, typically to form the nucleic acidduplex. Such time, temperature and pH conditions required to accomplishthe hybridization depend, as is well known in the art on the length ofthe polynucleotide probe to be hybridized, the degree of complementaritybetween the polynucleotide probe and the target, the guanidine andcytosine content of the polynucleotide, the stringency of thehybridization desired, and the presence of salts or additional reagentsin the hybridization reaction admixture as may affect the kinetics ofhybridization. Methods for optimizing hybridization conditions for agiven hybridization reaction admixture are well known in the art.

As used herein, the term “injury or neural injury” is intended toinclude a damage which directly or indirectly affects the normalfunctioning of the CNS or PNS. For example, the injury can be damage toretinal ganglion cells; a traumatic brain injury; a stroke relatedinjury; a cerebral aneurism related injury; demyelinating diseases suchas multiple sclerosis; a spinal cord injury, including monoplegia,diplegia, paraplegia, hemiplegia and quadriplegia; a neuroproliferativedisorder or neuropathic pain syndrome. Examples of CNS injuries ordisease include TBI, stroke, concussion (including post-concussionsyndrome), cerebral ischemia, neurodegenerative diseases of the brainsuch as Parkinson's disease, Dementia Pugilistica, Huntington's diseaseand Alzheimer's disease, brain injuries secondary to seizures which areinduced by radiation, exposure to ionizing or iron plasma, nerve agents,cyanide, toxic concentrations of oxygen, neurotoxicity due to CNSmalaria or treatment with anti-malaria agents, malaria pathogens, injurydue to trypanosomes, and other CNS traumas. Examples of PNS injuries ordiseases include neuropathies induced either by toxins (e.g. cancerchemotherapeutic agents) diabetes, peripheral trauma or any process thatproduced pathological destruction of peripheral nerves and/or theirmyelin sheaths.

As used herein, the term “stroke” is art recognized and is intended toinclude sudden diminution or loss of consciousness, sensation, andvoluntary motion caused by rapture or obstruction (e.g. by a blood clot)of an artery of the brain.

As used herein, the term “Traumatic Brain Injury” is art recognized andis intended to include the condition in which, a traumatic blow to thehead causes damage to the brain, often without penetrating the skull.Usually, the initial trauma can result in expanding hematoma,subarachnoid hemorrhage, cerebral edema, raised intracranial pressure(ICP), and cerebral hypoxia, which can, in turn, lead to severesecondary events due to low cerebral blood flow (CBF).

“Neural cells” as defined herein, are cells that reside in the brain,central and peripheral nerve systems, including, but not limited to,nerve cells, glial cell, oligodendrocyte, microglia cells or neural stemcells.

“Neuronal specific or neuronally enriched proteins” are defined herein,as proteins that are present in neural cells and not in non-neuronalcells, such as, for example, cardiomyocytes, myocytes, in skeletalmuscles, hepatocytes, kidney cells and cells in testis. Non-limitingexamples of neural proteins from which peptides can be derived via, forexample, enzyme degradation, are shown in Table 1 below.

“Neural (neuronal) defects, disorders or diseases” as used herein refersto any neurological disorder, including but not limited toneurodegenerative disorders (Parkinson's; Alzheimer's) or autoimmunedisorders (multiple sclerosis) of the central nervous system; memoryloss; long term and short term memory disorders; learning disorders;autism, depression, benign forgetfulness, childhood learning disorders,close head injury, and attention deficit disorder; autoimmune disordersof the brain, neuronal reaction to viral infection; brain damage;depression; psychiatric disorders such as bi-polarism, schizophrenia;narcolepsy/sleep disorders (including circadian rhythm disorders,insomnia and narcolepsy); severance of nerves or nerve damage; severanceof the cerebrospinal nerve cord (CNS) and any damage to brain or nervecells; neurological deficits associated with AIDS; tics (e.g. Giles dela Tourette's syndrome); Huntington's chorea, schizophrenia, traumaticbrain injury, tinnitus, neuralgia, especially trigeminal neuralgia,neuropathic pain, inappropriate neuronal activity resulting inneurodysthesias in diseases such as diabetes, MS and motor neurondisease, ataxias, muscular rigidity (spasticity) and temporomandibularjoint dysfunction; Reward Deficiency Syndrome (RDS) behaviors in asubject.

As used herein, “RDS” behaviors are those behaviors that manifests asone or more behavioral disorders related to an individual's feeling ofwell-being with anxiety, anger or a craving for a substance. RDSbehaviors include, alcoholism, SUD, smoking, BMI or obesity,pathological gambling, carbohydrate bingeing, axis 11 diagnosis, SAB,ADD/ADHD, CD, TS, family history of SUD, and Obesity. All thesebehaviors, and others described herein as associated with RDS behaviorsor genes involved in the neurological pathways related to RDS, areincluded as RDS behaviors as part of this invention. Additionally, manyof the clinical terms used herein for many specific disorders that areRDS disorders are found in the Quick Reference to the DiagnosticCriteria From DSM-IV™, The American Psychiatric Association, Washington,D.C., 1994.

Affective disorders, including major depression, and the bipolar,manic-depressive illness, are characterized by changes in mood as theprimary clinical manifestation. Major depression is the most common ofthe significant mental illnesses, and it must be distinguishedclinically from periods of normal grief, sadness and disappointment, andthe related dysphoria or demoralization frequently associated withmedical illness. Depression is characterized by feelings of intensesadness, and despair, mental slowing and loss of concentration,pessimistic worry, agitation, and self-deprecation. Physical changes canalso occur, including insomnia, anorexia, and weight loss, decreasedenergy and libido, and disruption of hormonal circadian rhythms.

Mania, as well as depression, is characterized by changes in mood as theprimary symptom. Either of these two extremes of mood may be accompaniedby psychosis with disordered thought and delusional perceptions.Psychosis may have, as a secondary symptom, a change in mood, and it isthis overlap with depression that causes much confusion in diagnosis.Severe mood changes without psychosis frequently occur in depression andare often accompanied by anxiety.

Parkinson's disease, independent of a specific etiology, is a chronic,progressive central nervous system disorder which usually appearsinsidiously in the latter decades of life. The disease produces a slowlyincreasing disability in purposeful movement. It is characterized byfour major clinical features of tremor, bradykinesia, rigidity and adisturbance of posture. Often patients have an accompanying dementia. Inidiopathic Parkinsonism, there is usually a loss of cells in thesubstantia nigra, locus ceruleus, and other pigmented neurons of thebrain, and a decrease of dopamine content in nerve axon terminals ofcells projecting from the substantia nigra. The understanding thatParkinsonism is a syndrome of dopamine deficiency and the discovery oflevodopa as an important drug for the treatment of the disease were thelogical culmination of a series of related basic and clinicalobservations, which serves as the rationale for drug treatment.

The term “Alzheimer's Disease” refers to a progressive mentaldeterioration manifested by memory loss, confusion and disorientationbeginning in late middle life and typically resulting in death in fiveto ten years. Pathologically, Alzheimer's Disease can be characterizedby thickening, conglutination, and distortion of the intracellularneurofibrils, neurofibrillary tangles and senile plaques composed ofgranular or filamentous argentophilic masses with an amyloid core.Diagnosing Alzheimer's Disease: the National Institute of Neurologicaland Communicative Disorders and Stroke-Alzheimer's Disease and theAlzheimer's Disease and Related Disorders Association (NINCDS-ADRDA)criteria can be used to diagnose Alzheimer's Disease (McKhann et al.,1984, Neurology 34:939-944). The patient's cognitive function can beassessed by the Alzheimer's Disease Assessment Scale-cognitive subscale(ADAS-cog; Rosen et al., 1984, Am. J. Psychiatry 141:1356-1364).

As used herein, the term “autism” refers to a state of mentalintroversion characterized by morbid self-absorption, social failure,language delay, and stereotyped behavior.

As used herein, the term “depression” refers to a clinical syndrome thatincludes a persistent sad mood or loss of interest in activities, whichlasts for at least two weeks in the absence of treatment.

The term “benign forgetfulness,” as used herein, refers to a mildtendency to be unable to retrieve or recall information that was onceregistered, learned, and stored in memory (e.g., an inability toremember where one placed one's keys or parked one's car). Benignforgetfulness typically affects individuals after 40 years of age andcan be recognized by standard assessment instruments such as theWechsler Memory Scale (Russell, 1975, J. Consult Clin. Psychol.43:800-809).

As used herein, the term “childhood learning disorders” refers to animpaired ability to learn, as experienced by certain children.

The term “close head injury,” as used herein, refers to a clinicalcondition after head injury or trauma which condition can becharacterized by cognitive and memory impairment.

The term “attention deficit disorder,” as used herein, refers to adisorder that is most commonly exhibited by children and which can becharacterized by increased motor activity and a decreased attentionspan. Attention-deficit disorder (“ADD”) is a common behavioral learningdisorder in children which adversely affects school performance andfamily relationships. Symptoms and signs include hyperactivity (e.g.,ADDH and AD/HD, DSM-IV), impulsivity, emotional ability, motorincoordination and some perceptual difficulties. Treatment has includedpsychostimulants, which while effective are controversial, and may causetroubling side effects such as dysphoria, headache and growthretardation. Other drugs, including the tricyclic antidepressants,appear to improve attention, but may be less effective than thepsychostimulants.

As used herein, “subcellular localization” refers to defined subcellularstructures within a single nerve cell. These subcellularly definedstructures are matched with unique neural proteins derived from, forexample, dendritic, axonal, myelin sheath, presynaptic terminal andpostsynaptic locations. By monitoring the release of peptides unique toeach of these regions, one can therefore monitor and define subcellulardamage after brain injury. Furthermore, mature neurons aredifferentiated into dedicated subtype fusing a primary neuraltransmitter such as cholinergic (nicotinic and mucarinic),glutamatergic, gabaergic, serotonergic, dopaminergic. Each of thisneuronal subtype express unique neural proteins such as those dedicatedfor the synthesis, metabolism and transporter and receptor of eachunique neurotransmitter system (Table 1 below).

As used herein, a “pharmaceutically acceptable” component is one that issuitable for use with humans and/or animals without undue adverse sideeffects (such as toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio.

The terms “patient” or “individual” are used interchangeably herein, andis meant a mammalian subject to be treated, with human patients beingpreferred. In some cases, the methods of the invention find use inexperimental animals, in veterinary application, and in the developmentof vertebrate models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters; birds, fish reptiles, and primates.

As used herein, “ameliorated” or “treatment” refers to a symptom whichis approaches a normalized value, e.g., is less than 50% different froma normalized value, preferably is less than about 25% different from anormalized value, more preferably, is less than 10% different from anormalized value, and still more preferably, is not significantlydifferent from a normalized value as determined using routinestatistical tests. For example, amelioration or treatment of depressionincludes, for example, relief from the symptoms of depression whichinclude, but are not limited to changes in mood, feelings of intensesadness and despair, mental slowing, loss of concentration, pessimisticworry, agitation, and self-deprecation. Physical changes may also berelieved, including insomnia, anorexia and weight loss, decreased energyand libido, and the return of normal hormonal circadian rhythms. Anotherexample, when using the terms “treating Parkinson's disease” or“ameliorating” as used herein means relief from the symptoms ofParkinson's disease which include, but are not limited to tremor,bradykinesia, rigidity, and a disturbance of posture.

Proteolytic Markers

This disclosure describes the novel and highly practical use ofproteolytic markers that can be detected in tissues, blood, cerebralspinal fluid (CSF) and other biological fluids (sweat, urine, saliva)for purposes of diagnosis and treatment following organ injury or tumor.Proteases are uniquely activated when cells are injured, stressed orchemically challenged. The over-activation of these proteases oftencontributes to cell death phenotypes, including apoptosis and oncosis(or oncotic necrosis) (see for example, FIG. 1). For instance, followingtraumatic brain injury (TBI), stroke and renal ischemia, calpains I andII become activated and, as a result, contribute to oncotic andapoptotic cell death. As well, activated caspases 3, 8 and 9 promoteapoptosis in these same disease conditions. In fact, there are manyproteases that are activated following organ injury, some of whichinclude cathepsin B, L, and D, MMP2, 9, and 13, UCH-L1, ubiquitinbinding proteases (UBP'S), chymase, tryptase and proteasome subunits(See Table 1). Table 1 shows non-limiting examples of potentialproteolytic enzymes and protease-sensitive tissue protein markers. Eachtissue protein marker in Table 1 can produce a proteolytic biomarkerwhen cleaved by enzymes. Table 2 below shows non-limiting examples ofunobvious and unique tissue protein cleavage sites produced by proteaseattack. For example, In a preferred embodiment, the invention providesbiomarkers that are indicative of traumatic brain injury, neuronaldamage, neural disorders, brain damage, neural damage due to drug oralcohol addiction, diseases associated with the brain or nervous system,such as the central and peripheral nervous systems (CNS, PNS).Preferably, the biomarkers are proteolytic enzymes which are activatedas a result of damage to organs such as for example: heart, brain,liver, kidneys, lung, gut; neurons, central nervous system, peripheralnervous system, as well as skeletal muscles. Preferably the proteolyticenzymes are activated and cleave target proteins, peptides and fragmentsthereof due to neural and organ injury. Target proteins include, but arenot limited to proteins, peptides or fragments thereof associated withneuronal cells, brain cells or any cell that is present in the brain andcentral nervous systems, organs such as heart, liver, kidneys and thelike. Non-limiting examples of proteolytic enzymes that are detectedupon neural and/or organ injury include (in alphabetical order):Achromopeptidase, Aminopeptidase, Ancrod, Angiotensin Converting Enzyme,Bromelain, Calpain, Calpain I, Calpain II, Carboxypeptidase A,Carboxypeptidase B, Carboxypeptidase G, Carboxypeptidase P,Carboxypeptidase W, Carboxypeptidase Y, Caspase, Caspase 1, Caspase 2,Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8,Caspase 9, Caspase 10, Caspase 13, Cathepsin B, Cathepsin C, CathepsinD, Cathepsin G, Cathepsin H, Cathepsin L, Chymopapain, Chymase,Chymotrypsin, α-Clostripain, Collagenase, Complement C1r, ComplementC1s, Complement Factor D, Complement factor I, Cucumisin, Dipeptidylpeptidase IV, Elastase, leukocyte, Elastase, pancreatic, EndoproteinaseArg-C, Endoproteinase Asp-N, Endoproteinase Glu-C, Endoproteinase Lys-C,Enterokinase, Factor Xa, Ficin, Furin, Granzyme A, Granzyme B, HIVProtease, Igase, Kallikrein tissue, Kinase, Leucine Aminopeptidase(General), Leucine aminopeptidase, cytosol, Leucine aminopeptidase,microsomal, Matrix metalloprotease, Methionine Aminopeptidase, Neutrase,Papain, Pepsin, Plasmin, Prolidase, Pronase E, Prostate SpecificAntigen, Protease, Protease S, Proteasomes, Proteinase, Proteinase 3,Proteinase A, Proteinase K, Protein C, Pyroglutamate aminopeptidase,Renin, Rennin, Thrombin, Tissue Plasminogen Activator, Troponins,Trypsin, Tryptase, Urokinase. Preferably, any one of SEQ ID NO's.: 1-148are also detected.

By tracking the over-activation of these, and other proteases, one coulddiagnose and aid in the therapeutic treatment of organ diseases,including, but not limited to, stroke or brain injury, renal failure,lung disease, heart attack and cataract formation. In case of canceroustumors, there is likely to be increased cell death inside an activetumor, due to its rapid growth. Cell death is also significantlyelevated during cancer treatment (e.g. chemotherapy) where the objectiveis to induce apoptosis of tumor cells. Thus, measuring proteaseactivation (as a cell death index) would be a useful tool in trackingthe progress of tumor growth and success of certain therapeutictreatments in treating cancer.

Two approaches to measure disease or medically induced proteaseactivation will be used. The first is to track the activation ofproteolytic enzymes directly. Because most proteases undergo proteolyticprocessing before becoming fully activated, truncation sites can beidentified. With this knowledge, one could build specific tools todetect their activation, for example, we have already employed this typeof technology with the use of an anti-activated calpain I antibody. Thistool is particularly powerful if a protease is specifically or highlyexpressed in a distinct organ of interest. The second technologicalapproach is to examine substrates that are cleaved by activatedproteases (see Table 1). For instance, activated calpains cleave severalproteins, including β1-spectrin, β1-spectrin, MAP2A/2B, synaptotagmin,tau, neurofilament H, M, and L and myelin basic protein. Armed with theknowledge of exact substrate cleavage sites, fragment-specificantibodies can be developed. Again, the power of this technique isparticularly notable when a known tissue-specific substrate is cleaved,because this cleavage product can serve as a biomarker for that tissuetype.

The advantages to measuring proteolytic markers in disease conditionsare three-fold. I) The concept of excessive protease activation is acommon theme in cancer and in many tissue and organ injuries, including,but not limited to, the brain, liver kidney, and heart. II) Manyproteolytic products of activated proteases are released into biologicalfluids such blood, CSF, urine, sweat and saliva. Although theirconcentrations would be lower than the levels found directly within theoriginating injured tissue, they could still be detected (usingantibodies or other capture agents), quantified and correlated withother outcome measures. III) The ability to use relatively non-invasiveprocedures to diagnose, treat and track patients is another powerfulutility to using proteolytic markers in disease conditions.

In a preferred embodiment, detection of proteolytic enzymes that degradeone or more cleavage products is diagnostic of neural damage and/orneuronal disease. Examples of substrates of detected proteolytic enzymesinclude but are not limited to neural peptides, such as for example,axonal peptides—NF-200 (NF-H), NF-160 (NF-M), NF-68 (NF-L); amyloidprecursor peptides; dendritic peptides—alpha-tubulin (P02551),beta-tubulin (P0 4691), MAP-2A/B, MAP-2C, Tau, Dynamin-1 (P21575),Dynactin (Q13561), P24; somal peptides—UCH-L1 (Q00981), PEBP (P31044),NSE (P07323), Thy 1.1, Prion, Huntington; presynapticpeptides—synapsin-1, synapsin-2, alpha-synuclein (p37377),beta-synuclein (Q63754), GAP43, synaptophysin, synaptotagmin (P21707),syntaxin; post-synaptic peptides —PSD95, PSD93, NMDA-receptor (includingall subtypes); demyelination biomarkers—myelin basic peptides (MBP),myelin proteolipid peptides, glial peptides—GFAP (P47819), proteindisulfide isomerase peptides (PDI-P04785); neurotransmitterbiomarkers—cholinergic biomarkers: acetylcholine esterase peptides,choline acetyltransferase peptides; dopaminergic biomarkers—tyrosinehydroxylase peptides (TH), phospho-TH peptides, DARPP32 peptides;noradrenergic biomarkers—dopamine beta-hydroxylase peptides (DbH);serotonergic biomarkers—tryptophan hydroxylase peptides (TrH);glutamatergic biomarkers—glutaminase peptides, glutamine synthetasepeptides; GABAergic biomarkers—GABA transaminase peptides(4-aminobutyrate-2-ketoglutarate transaminase [GABAT]), glutamic aciddecarboxylase peptides (GAD25, 44, 65, 67); neurotransmitterreceptors—beta-adrenoreceptor subtype peptides, (e.g. beta (2)),alpha-adrenoreceptor subtype peptides, (e.g. (alpha (2c)), peptides ofGABA receptors (e.g. GABA (B)), peptides of metabotropic glutamatereceptor (e.g. mGluR3), NMDA receptor subunit peptides (e.g. NR1A2B),Glutamate receptor subunit peptides (e.g. GluR4), peptides of 5-HTserotonin receptors (e.g. 5-HT(3)), peptides of dopamine receptors (e.g.D4), peptides of muscarinic Ach receptors (e.g. M1), peptides ofnicotinic acetylcholine receptor (e.g. alpha-7); neurotransmittertransporters—peptides of norepinephrine transporter (NET), peptides ofdopamine transporter (DAT), peptides of serotonin transporter (SERT),vesicular transporter peptides (VMAT1 and VMAT2), peptides of GABAtransporter vesicular inhibitory amino acid transporter (VIAAT/VGAT),peptides of glutamate transporter (e.g. GLT1), peptides of vesicularacetylcholine transporter, peptides of choline transporter (e.g. CHT1);other peptide biomarkers include, but not limited to vimentin peptides(P31000), CK-BB peptides (P07335), 14-3-3-epsilon (P42655) peptides,MMP2 peptides, MMP9 peptides.

In another preferred embodiment, the proteolytic enzyme biomarkers havea specific activity for the neural proteins, for example the nonlimiting examples listed in Table 1, about 1 g to about 500 μg per 1 mgof substrate protein.

It has been shown experimentally that if the amount of the enzyme iskept constant and the substrate concentration is then graduallyincreased, the reaction velocity will increase until it reaches amaximum. After this point, increases in substrate concentration will notincrease the velocity (delta A/delta T). It is theorized that when thismaximum velocity had been reached, all of the available enzyme has beenconverted to ES, the enzyme substrate complex. This point on the graphis designated V_(max). Using this maximum velocity and equation (7),Michaelis developed a set of mathematical expressions to calculateenzyme activity in terms of reaction speed from measurable laboratorydata.

The Michaelis constant Km is defined as the substrate concentration at ½the maximum velocity. Using this constant and the fact that Km can alsobe defined as:

${Km} = {\frac{K_{+ 1} + K_{+ 2}}{K_{- 1}} = \lbrack S\rbrack_{V_{\frac{mx}{2}}}}$

K₊₁, K⁻¹ and K₊₂ being the rate constants from equation (7). Michaelisdeveloped the following

$V_{1} = \frac{V_{mx}\lbrack S\rbrack}{K_{m} + \lbrack S\rbrack}$

where

-   -   V_(†)=velocity at any time    -   [S]=the substrate concentration at this time    -   V_(max) =the highest under this set of experimental conditons        (pH, temperature etc.)    -   K_(m)=the Michaelis constant for the particular enzyme being        investigated

Michaelis constants have been determined for many of the commonly usedenzymes. A small Km indicates that the enzyme requires only a smallamount of substrate to become saturated. Hence, the maximum velocity isreached at relatively low substrate concentrations. A large Km indicatesthe need for high substrate concentrations to achieve maximum reactionvelocity.

The substrate with the lowest Km upon which the enzyme acts as acatalyst is frequently assumed to be enzyme's natural substrate, thoughthis is not true for all enzymes.

Without wishing to be bound by theory, upon injury, structural andfunctional integrity of the cell membrane and blood brain barrier arecompromised. Brain-specific and brain-enriched proteins, peptides orfragments thereof, are released into the extracellular space andsubsequently into the CSF and blood. Proteolytic enzymes specific forthese substrates are activated and cleave the substrate. Detection ofone or more of these proteolytic enzyme biomarkers is indicative ofneural and/or organ injury.

In a preferred embodiment, detection of at least one proteolytic enzymespecific for neural peptides released by injured neural cells and/ororgans in CSF, blood, or other biological fluids, is diagnostic of theseverity of brain injury and/or the monitoring of the progression oftherapy. Preferably, the proteolytic enzyme markers are detected duringthe early stages of injury. An increase in the amount of proteolyticenzyme biomarkers fragments or derivatives thereof, in a patientsuffering from a neural injury, neuronal disorder as compared to anormal healthy individual, will be diagnostic of a neural injury and/orneuronal disorder.

In a preferred embodiment, the invention provides biomarkers that areindicative of traumatic brain injury, neuronal damage to the CNS or PNS,neural disorders, brain damage, neural damage due to drug or alcoholaddiction, diseases associated with the brain or nervous system, such asthe central nervous system. Preferably, the biomarkers are proteolyticenzymes which are activated as a result of damage to organs such as forexample: heart, brain, liver, kidneys; neurons, central nervous system,peripheral nervous system. Preferably the proteolytic enzymes areactivated and cleave target proteins, peptides and fragments thereof dueto neural and organ injury. Target proteins include, but are not limitedto proteins, peptides or fragments thereof associated with neuronalcells, brain cells or any cell that is present in the brain and centralnervous system, organs such as heart, liver, kidneys and the like.Non-limiting examples of proteolytic enzymes that are detected uponneural and/or organ injury include (in alphabetical order):Achromopeptidase, Aminopeptidase, Ancrod, Angiotensin Converting Enzyme,Bromelain, Calpain, Calpain I, Calpain II, Carboxypeptidase A,Carboxypeptidase B, Carboxypeptidase G, Carboxypeptidase P,Carboxypeptidase W, Carboxypeptidase Y, Caspase, Caspase 1, Caspase 2,Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8,Caspase 9, Caspase 10, Caspase 13, Cathepsin B, Cathepsin C, CathepsinD, Cathepsin G, Cathepsin H, Cathepsin L, Chymopapain, Chymase,Chymotrypsin, α-Clostripain, Collagenase, Complement C1r, ComplementC1s, Complement Factor D, Complement factor I, Cucumisin, Dipeptidylpeptidase IV, Elastase, leukocyte, Elastase, pancreatic, EndoproteinaseArg-C, Endoproteinase Asp-N, Endoproteinase Glu-C, Endoproteinase Lys-C,Enterokinase, Factor Xa, Ficin, Furin, Granzyme A, Granzyme B, HIVProtease, Igase, Kallikrein tissue, Kinase, Leucine Aminopeptidase(General), Leucine aminopeptidase, cytosol, Leucine aminopeptidase,microsomal, Matrix metalloprotease, Methionine Aminopeptidase, Neutrase,Papain, Pepsin, Plasmin, Prolidase, Pronase E, Prostate SpecificAntigen, Protease, Protease S, Proteasomes, Proteinase, Proteinase 3,Proteinase A, Proteinase K, Protein C, Pyroglutamate aminopeptidase,Renin, Rennin, Thrombin, Tissue Plasminogen Activator, Troponins,Trypsin, Tryptase, Urokinase.

In another preferred embodiment, detection of at least one proteolyticenzyme, which has a neural peptide as a substrate, in CSF, blood, orother biological fluids, is diagnostic of the severity of injuryfollowing a variety of CNS insults, such as for example, stroke, spinalcord injury, or neurotoxicity caused by alcohol or substance abuse (e.g.ecstasy, methamphetamine, etc.).

The CNS comprises many brain-specific and brain-enriched peptides thatare preferable substrates for proteolytic enzyme biomarkers in thediagnosis of brain injury, neural injury, neural disorders fragments andderivatives thereof. Non-limiting examples of substrates for proteolyticenzymes are shown in Table 1. Table 2 shows non-limiting examples ofunobvious and unique tissue protein cleavage sites produced by proteaseattack. (For example, SEQ ID NO's.: 1-148). For example, the proteolyticenzyme biomarkers are specific for neural specific proteins and caninclude axonal peptides such as neurofilament-heavy (NF-200),neurofilament-medium (NF-160), neurofilament-light (NF-68), and amyloidprecursor peptides; dendritic peptides such as alpha-tubulin,beta-tubulin, MAP-2A/B/C. tau, dynamin-1, dynactin; and peptides foundin the soma (cell body) including ubiquitin C-terminal hydrolase L1peptides (UCH-L1), PEBP peptides, neuronal-specific enolase peptides(NSE), NeuN peptides, Thy 1.1 peptides, Prion and Huntington peptides.There are also peptides found pre-synaptically and post-synaptically.Moreover, different types of neurons exhibit distinctneurotransmitter-specific enzyme pathway proteins from which peptidesare identified. For example, acetylcholine esterase is found only incholinergic neurons while tyrosine hydroxylase (TII) is exclusive todopaminergic neurons. Other neurotransmitter-specific enzyme pathwaypeptides include dopamine beta hydroxylase peptides (DbH) innoradrenergic neurons, tryptophan hydroxylase peptides (TrH) inserotonergic neurons, peptides of glutaminase and glutamine synthetasein glutamatergic neurons, and GABA transaminase peptides and glutamicacid decarboxylase peptides in GABAergic neurons. Furthermore, peptidesfrom proteins such as GFAP and protein disulfide isomerase (PDI) areonly synthesized in glial cells of the CNS, a feature that could beexploited to further understand the extent of damage to the CNS.Therefore, detection of one or more proteolytic enzyme biomarkers isindicative of the cell injured, the severity of injury and the type ofinjury. For example, tumors shed antigens, activation of proteolyticenzymes specific for these antigens is diagnostic of a tumor and type oftumor.

In another preferred embodiment, the invention provides for thequantitative detection of damage to the CNS, PNS and/or brain injury ata subcellular level. Depending on the type and severity of injury,neurons can undergo damage in specific cellular regions. For example,proteolytic enzymes specific for certain polypeptides, such as forexample, axonal peptides, fragments and derivatives thereof include, butnot limited to: peptides of NF-200 (NF-H), NF-160 (NF-M), NF-68 (NF-L),fragments and derivatives thereof, differentiates between axonal versusdendritic damage. Non-limiting examples of substrates for proteolyticenzyme biomarkers, such as dendritic peptides, fragments and derivativesthereof, include, but not limited to: alpha-tubulin (P02551),beta-tubulin (P0 4691), MAP-2A/B, MAP-2C, Tau, Dynamin-1 (P21575),Dynactin (Q13561), P24. Furthermore, detection of different biomarkersnot only differentiate between, for example, axonal or dendritic damage,but allow for the assessment of synaptic pathology, specific injury toelements of the pre-synaptic terminal and post-synaptic density.

In another preferred embodiment, detection of certain proteolytic enzymebiomarkers are diagnostic of the specific cell type affected followinginjury since neurons and glia possess distinct proteins. For example,proteolytic enzymes specific for glial proteins, peptides, fragments andderivatives thereof is diagnostic of glial cell damage. Examples ofglial peptides, include, but not limited to: peptides of GFAP (P47819),protein disulfide isomerase peptides (PDI-P04785).

The ability to detect and monitor levels of these proteolytic enzymebiomarkers after CNS injury provides enhanced diagnostic capability byallowing clinicians (1) to determine the level of injury severity inpatients with various CNS injuries, (2) to monitor patients for signs ofsecondary CNS injuries that may elicit these cellular changes and (3) tomonitor the effects of therapy by examination of these peptides in CSFor blood. Unlike other organ-based diseases where rapid diagnostics forsurrogate biomarkers prove invaluable to the course of action taken totreat the disease, no such rapid, definitive diagnostic tests exist fortraumatic or ischemic brain injury that might provide physicians withquantifiable neurochemical markers to help determine the seriousness ofthe injury, the anatomical and cellular pathology of the injury, and theimplementation of appropriate medical management and treatment.

In comparison to currently existing products, the invention providesseveral superior advantages and benefits. First, the identification ofneuronal biomarkers provide more rapid and less expensive diagnosis ofinjury severity than existing diagnostic devices such as computedtomography (CT) and magnetic resonance imaging (MRI). The invention alsoallows quantitative detection and high content assessment of damage tothe CNS at a subcellular level (i.e. axonal versus dendritic). Theinvention also allows identification of the specific cell type affected(for example, neurons versus glia). In addition, levels of thesebrain-specific and brain-enriched peptides provides more accurateinformation regarding the level of injury severity than what is on themarket.

In another preferred embodiment, nerve cell damage in a subject isanalyzed by (a) providing a biological sample isolated from a subjectsuspected of having a damaged nerve cell; (b) detecting in the samplethe presence or amount of at least one marker selected from one or moreneural proteins; and (c) correlating the presence or amount of themarker with the presence or type of nerve cell damage in the subject.

Detection and identification of proteolytic enzyme biomarkers aredetectable by various methods known in the art. For example, fluorogenicassays or colorimetric assays. Assays for detection of different enzymesare commercially available. An example is the fluorogenic assay fromProteus BioSciences Inc. (San Diego, Calif.), Sigma (St. Louis, Mo.). Anexample for fluorescent detection of active caspase-1 protein. AFC(7-amino-4-trifluoromethylcoumarin*) is a synthetic fluorogenic compoundthat is hydrolyzed by the enzyme and yields a product that can bemeasured using a fluorimeter or spectrophotometer. The plate can be readat A₃₈₀ for chromogenic or Em₅₁₀₋₅₄₀ (Excitation at 390-400 nm) forfluorogenic detection

Any animal that expresses produces proteolytic enzymes are preferred.Preferably, the subject is a mammal, such as for example, a human, dog,cat, horse, cow, pig, sheep, goat, primate, rat, or mouse; vertebratessuch as birds, fish and reptiles. More preferably, the subject is ahuman. Particularly preferred are subjects suspected of having or atrisk for developing traumatic or non-traumatic nervous system injuries,such as victims of brain injury caused by traumatic insults (e.g.gunshots wounds, automobile accidents, sports accidents, shaken babysyndrome), ischemic events (e.g. stroke, cerebral hemorrhage, cardiacarrest), neurodegenerative disorders (such as Alzheimer's, Huntington's,and Parkinson's diseases; Prion-related disease; other forms ofdementia), epilepsy, substance abuse (e.g., from amphetamines,Ecstasy/MDMA, or ethanol), and peripheral nervous system pathologiessuch as diabetic neuropathy, chemotherapy-induced neuropathy andneuropathic pain.

TABLE 1 Examples of novel tissue proteins vulnerable to proteolyticattack Truncated forms of biomarkers Rat (mouse) accession (based onPowerblot data) Human accession number number a/b-SNAP P54920, Q9H115P54921 Adaptin P17426 AKAP220 Q9UKA4 Q62924 Alpha and beta-tubulinsP02551, P04691 Q68FR8, P24636 Alpha synuclein P37840 P37377 AmphiphysinP49418 O08838 Arp3 P61158 Q99J72(mouse) ASAP1 (ARF GTPase-activatingQ9ULH1 Q9QWY8(mouse) protein) ATP Synthase a P15999 Bad Q92934 O35147Bax (tBax) Q07812, Q07814 Q63690 Bcl2(tBcl2) P10415 P49950BetaIII-spectrin O15020 NP_062040 BetaII-Spectrin Q01082 BRaf P15056BRMP2 O08539 cAIP's AAN23755 CalgranulinB B31848 Calmodulin dependentkinase CAI13791, Q5SQZ3, Q13554 P11275 Calpastatin NP_001741 NP_445747CaMPKII CAI13791, Q13554 P11275, NP_037052 CaMPKIV NP_001735 NP_036859Catenin/pp120 P30999 βCatenin P35222 Q9WU82 Cathepsin L CAI16307 cCblQ16773 Q08415 Clathrin Heavy Chain P11442 Cofilin P23528 P45592collagen, type IV, alpha 6 AAP35892 CtBP1 Q13363 Q9Z2F5 DRBP76 Q12906Dynactin Q14203, Q13561, O00399 P28023, Q6AYH5 Dynamin Q05193, P50570,Q9UQ16 P21575, P39052, Q08877 Endopeptidase P42676 Fibronectin, O088871GABABR2 O75899 O88871 Glutamate (AMPA/Kainate) receptors NP_000818NP_113796 GlycogenphosphorylaseBB P11216 P53534 GSPT2 Q8IYD1NP_032205mouse hPrp17 O60508 Q8BJF8mouse Huntingtin P42858 Integrinbeta3 P05106 Ki67 P46013 Lamin A, B, C P02545; Laminin MCalherin MEF2DQ14814 Q66HL8 Metabotropic glutamate recpetors NP_000829, NP_000832NP_058707, NP_073157 mGluR1 Q13255 P23385 Munc18 Q02410, Q99767, O96018Myelin basic protein (tMBP) MBHUB P02686 Myelin Oligodendrocyte specificAAC25187 protein(MOSP) Myosin light chain O14950 P02600 MYPT1 O14974Q10728 NCK P16333, O43639 Nek2 P51955 Q91XQ1 Neuronal protein 22(NP22);transgelin-3 NP_037391 AAL66341 Neuronal protein 25(NP25) AAP97165NP_113864 NMDAR1 (NR1) NP_067544 NP_058706 AAB29181; P35439| NMDAR2(NR2A, 2B, 2C, 2D) Q5IS45; NP_000825; Q00959; Q00960; NP_000826;NP_000827 NMDAR3, NMDAR 4 (NR3, NR4) AAB60368 Q9R1M7| Q91ZU9 Neuronalnitric oxide synthase (nNOS) G01946 NP_032738 N-ethylmaleimide sensitivefusion P46459 NP_542152 protein (NSF) Nucleoporin NSP1 Q9Y2X4 AAB33384P150Glued P28023 AAB24566 Rho-GTPase-activating protein 5 (p190- Q13017P84107 B) P55Cdc Q12834 Q62623 PMCA2 Q01814 P11506 Profilin P07737,P35080, P60673, Q8NHR9 P62963, Q9EPC6 PSD93 — AAC52643 Rabphilin3AQ9Y2J0 P47709 Macrophage-stimulating protein Q04912 — receptor precursor(RONa) Wiskott-Aldrich syndrome protein family Q92558 — member 1 (SCAR1)Transcription elongation factor S-II P23193 Q63799 protein 1 (SII/TFIIS)Smac/Diablo Q9NR28 Q9JIQ3mouse SNAP25 P60880 P60881 Striatin O43815P70483 Synapsin I P17600 P09951 Synapsin II Q92777 Q63537 Synapsin IIIO14994 NP_038750 Synaptojanin-I, II Q62910, O15056; O43426 Q62910,O55207 Synaptotagmin-I P21579 P21707 TNIK Q9UKE5 P83510mouse αPKC-P17252 P05696 βPkC- P05771 P68403 εPKC- P24723 Q64617 γPKC- P05129P63319

As described above, the invention provides the step of correlating thepresence or amount of one or more proteolytic enzyme biomarker(s) withthe severity and/or type of nerve cell injury. In a preferredembodiment, detection of a proteolytic enzyme biomarker can becorrelated with the presence of substrate protein for which thebiomarker a specific activity preferably from about 1 μg to about 500 μgper 1 mg of substrate protein per being proteolyzed in injured orstressed organs (in vivo) within minutes to days after or in vitro usingpurified protease-substrate protein/protein mixture ratio of 1/10,000 to1/20 at a time point within minutes to hours. For example, the amount ofneural peptides directly relates to severity of nerve tissue injury asmore severe injury damages a greater number of nerve cells which in turncauses a larger amount of neural peptide(s) to accumulate in thebiological sample (e.g., CSF). Whether a nerve cell injury triggers anapoptotic, oncotic (necrotic) or type 2 (autophagic) cell death, can bedetermined by examining the unique proteolytic enzyme biomarkers whichhave a high specific activity for peptides released into the biofluid inresponse to different cell death phenotype. The peptides can also bedetected from the many cell types that comprise the nervous system. Forexample, astroglia, oligodendrocytes, microglia cells, Schwann cells,fibroblast, neuroblast, neural stem cells and mature neurons.Furthermore, mature neurons are differentiated into dedicated subtypefusing a primary neural transmitter such as cholinergic (nicotinic andmucarinic), glutamatergic, gabaergic, serotonergic, dopaminergic. Eachof this neuronal subtype express unique neural proteins such as thosededicated for the synthesis, metabolism and transporter and receptor ofeach unique neurotransmitter system (Table 1). Lastly, within a singlenerve cell, there are subcellularly defined structures matched withunique neural proteins (dendritic, axonal, myelin sheath, presynapticterminal and postsynaptic density). By monitoring the release ofpeptides unique to each of these regions, subcellular damage can bemonitored and defined and correlated with the detection of proteolyticenzyme biomarkers after brain injury.

The biomarkers of the invention can be detected in a sample by anymeans. For example, immunoassays, include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,fluorescent immunoassays fragments and derivatives thereof. Such assaysare routine and well known in the art (see, e.g., Ausubel et al, eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York, which is incorporated by reference herein in itsentirety).

In another preferred embodiment, cardiac injury is determined by anincrease in cardiac Troponins such as for example, troponin I. Duringcardiac cell damage and death, cellular contents are released into theblood stream such as cardiac troponin I.

U.S. Pat. No. 5,795,725 entitled “Methods for the Assay of Troponin Iand T and Selection of Autoantibodies for use in Immunoassays” disclosesassays and antibodies for detection and quantitation of cardiac specificTroponin I and Troponin T in body fluids as an indicator of myocardialinfarction.

Identification of New Markers

In a preferred embodiment, a biological sample is obtained from apatient with neural injury. Biological samples comprising biomarkersfrom other patients and control subjects (i.e. normal healthyindividuals of similar age, sex, physical condition) are used ascomparisons. Biological samples are extracted as discussed above.Preferably, the sample is prepared prior to detection of biomarkers.Typically, preparation involves fractionation of the sample andcollection of fractions determined to contain the biomarkers. Methods ofpre-fractionation include, for example, size exclusion chromatography,ion exchange chromatography, heparin chromatography, affinitychromatography, sequential extraction, gel electrophoresis and liquidchromatography. The analytes also may be modified prior to detection.These methods are useful to simplify the sample for further analysis.For example, it can be useful to remove high abundance proteins, such asalbumin, from blood before analysis.

In one embodiment, a sample can be pre-fractionated according to size ofproteins in a sample using size exclusion chromatography. For abiological sample wherein the amount of sample available is small,preferably a size selection spin column is used. In general, the firstfraction that is eluted from the column (“fraction 1”) has the highestpercentage of high molecular weight proteins; fraction 2 has a lowerpercentage of high molecular weight proteins; fraction 3 has even alower percentage of high molecular weight proteins; fraction 4 has thelowest amount of large proteins; and so on. Each fraction can then beanalyzed by immunoassays, gas phase ion spectrometry, fragments andderivatives thereof, for the detection of markers.

In another embodiment, a sample can be pre-fractionated by anionexchange chromatography. Anion exchange chromatography allowspre-fractionation of the proteins in a sample roughly according to theircharge characteristics. For example, a Q anion-exchange resin can beused (e.g., Q HyperD F, Biosepra), and a sample can be sequentiallyeluted with eluants having different pH's. Anion exchange chromatographyallows separation of biomarkers in a sample that are more negativelycharged from other types of biomarkers. Proteins that are eluted with aneluant having a high pH is likely to be weakly negatively charged, and afraction that is eluted with an eluant having a low pH is likely to bestrongly negatively charged. Thus, in addition to reducing complexity ofa sample, anion exchange chromatography separates proteins according totheir binding characteristics.

In yet another embodiment, a sample can be pre-fractionated by heparinchromatography. Heparin chromatography allows pre-fractionation of themarkers in a sample also on the basis of affinity interaction withheparin and charge characteristics. Heparin, a sulfatedmucopolysaccharide, will bind markers with positively charged moietiesand a sample can be sequentially eluted with eluants having differentpH's or salt concentrations. Markers eluted with an eluant having a lowpH are more likely to be weakly positively charged. Markers eluted withan eluant having a high pH are more likely to be strongly positivelycharged. Thus, heparin chromatography also reduces the complexity of asample and separates markers according to their binding characteristics.

In yet another embodiment, a sample can be pre-fractionated by isolatingproteins that have a specific characteristic, e.g. are glycosylated. Forexample, a CSF sample can be fractionated by passing the sample over alectin chromatography column (which has a high affinity for sugars).Glycosylated proteins will bind to the lectin column andnon-glycosylated proteins will pass through the flow through.Glycosylated proteins are then eluted from the lectin column with aneluant containing a sugar, e.g., N-acetyl-glucosamine and are availablefor further analysis.

Thus there are many ways to reduce the complexity of a sample based onthe binding properties of the proteins in the sample, or thecharacteristics of the proteins in the sample.

In yet another embodiment, a sample can be fractionated using asequential extraction protocol. In sequential extraction, a sample isexposed to a series of adsorbents to extract different types ofbiomarkers from a sample. For example, a sample is applied to a firstadsorbent to extract certain proteins, and an eluant containingnon-adsorbent proteins (i.e., proteins that did not bind to the firstadsorbent) is collected. Then, the fraction is exposed to a secondadsorbent. This further extracts various proteins from the fraction.This second fraction is then exposed to a third adsorbent, and so on.

Any suitable materials and methods can be used to perform sequentialextraction of a sample. For example, a series of spin columns comprisingdifferent adsorbents can be used. In another example, a multi-wellcomprising different adsorbents at its bottom can be used. In anotherexample, sequential extraction can be performed on a probe adapted foruse in a gas phase ion spectrometer, wherein the probe surface comprisesadsorbents for binding biomarkers. In this embodiment, the sample isapplied to a first adsorbent on the probe, which is subsequently washedwith an eluant. Markers that do not bind to the first adsorbent areremoved with an eluant. The markers that are in the fraction can beapplied to a second adsorbent on the probe, and so forth. The advantageof performing sequential extraction on a gas phase ion spectrometerprobe is that markers that bind to various adsorbents at every stage ofthe sequential extraction protocol can be analyzed directly using a gasphase ion spectrometer.

In yet another embodiment, biomarkers in a sample can be separated byhigh-resolution electrophoresis, e.g., one or two-dimensional gelelectrophoresis. A fraction containing a marker can be isolated andfurther analyzed by gas phase ion spectrometry. Preferably,two-dimensional gel electrophoresis is used to generate two-dimensionalarray of spots of biomarkers, including one or more markers. See, e.g.,Jungblut and Thiede, Mass Spectr. Rev. 16:145-162 (1997).

The two-dimensional gel electrophoresis can be performed using methodsknown in the art. See, e.g., Deutscher ed., Methods In Enzymology vol.182. Typically, biomarkers in a sample are separated by, e.g.,isoelectric focusing, during which biomarkers in a sample are separatedin a pH gradient until they reach a spot where their net charge is zero(i.e., isoelectric point). This first separation step results inone-dimensional array of biomarkers. The biomarkers in one dimensionalarray is further separated using a technique generally distinct fromthat used in the first separation step. For example, in the seconddimension, biomarkers separated by isoelectric focusing are furtherseparated using a polyacrylamide gel, such as polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).SDS-PAGE gel allows further separation based on molecular mass ofbiomarkers. Typically, two-dimensional gel electrophoresis can separatechemically different biomarkers in the molecular mass range from1000-200,000 Da within complex mixtures.

Biomarkers in the two-dimensional array can be detected using anysuitable methods known in the art. For example, biomarkers in a gel canbe labeled or stained (e.g., Coomassie Blue or silver staining). If gelelectrophoresis generates spots that correspond to the molecular weightof one or more markers of the invention, the spot can be furtheranalyzed by densitometric analysis or gas phase ion spectrometry. Forexample, spots can be excised from the gel and analyzed by gas phase ionspectrometry. Alternatively, the gel containing biomarkers can betransferred to an inert membrane by applying an electric field. Then aspot on the membrane that approximately corresponds to the molecularweight of a marker can be analyzed by gas phase ion spectrometry. In gasphase ion spectrometry, the spots can be analyzed using any suitabletechniques, such as MALDI or SELDI.

Prior to gas phase ion spectrometry analysis, it may be desirable tocleave biomarkers in the spot into smaller fragments using cleavingreagents, such as proteases (e.g., trypsin). The digestion of biomarkersinto small fragments provides a mass fingerprint of the biomarkers inthe spot, which can be used to determine the identity of markers ifdesired.

In yet another embodiment, high performance liquid chromatography (HPLC)can be used to separate a mixture of biomarkers in a sample based ontheir different physical properties, such as polarity, charge and size.HPLC instruments typically consist of a reservoir of mobile phase, apump, an injector, a separation column, and a detector. Biomarkers in asample are separated by injecting an aliquot of the sample onto thecolumn. Different biomarkers in the mixture pass through the column atdifferent rates due to differences in their partitioning behaviorbetween the mobile liquid phase and the stationary phase. A fractionthat corresponds to the molecular weight and/or physical properties ofone or more markers can be collected. The fraction can then be analyzedby gas phase ion spectrometry to detect markers.

Optionally, a marker can be modified before analysis to improve itsresolution or to determine its identity. For example, the markers may besubject to proteolytic digestion before analysis. Any protease can beused. Proteases, such as trypsin, that are likely to cleave the markersinto a discrete number of fragments are particularly useful. Thefragments that result from digestion function as a fingerprint for themarkers, thereby enabling their detection indirectly. This isparticularly useful where there are markers with similar molecularmasses that might be confused for the marker in question. Also,proteolytic fragmentation is useful for high molecular weight markersbecause smaller markers are more easily resolved by mass spectrometry.In another example, biomarkers can be modified to improve detectionresolution. For instance, neuraminidase can be used to remove terminalsialic acid residues from glycoproteins to improve binding to an anionicadsorbent and to improve detection resolution. In another example, themarkers can be modified by the attachment of a tag of particularmolecular weight that specifically bind to molecular markers, furtherdistinguishing them. Optionally, after detecting such modified markers,the identity of the markers can be further determined by matching thephysical and chemical characteristics of the modified markers in aprotein database (e.g., SwissProt, MASCOT).

After preparation, biomarkers in a sample are typically captured on asubstrate for detection. Traditional substrates include antibody-coated96-well plates or nitrocellulose membranes that are subsequently probedfor the presence of proteins. Preferably, the biomarkers are identifiedusing immunoassays as described above. However, preferred methods alsoinclude the use of biochips. Preferably the biochips are proteinbiochips for capture and detection of proteins. Many protein biochipsare described in the art. These include, for example, protein biochipsproduced by Packard BioScience Company (Meriden Conn.), Zyomyx (Hayward,Calif.) and Phylos (Lexington, Mass.). In general, protein biochipscomprise a substrate having a surface. A capture reagent or adsorbent isattached to the surface of the substrate. Frequently, the surfacecomprises a plurality of addressable locations, each of which locationhas the capture reagent bound there. The capture reagent can be abiological molecule, such as a polypeptide or a nucleic acid, whichcaptures other biomarkers in a specific manner. Alternatively, thecapture reagent can be a chromatographic material, such as an anionexchange material or a hydrophilic material. Examples of such proteinbiochips are described in the following patents or patent applications:U.S. Pat. No. 6,225,047 (Hutchens and Yip, “Use of retentatechromatography to generate difference maps,” May 1, 2001), Internationalpublication WO 99/51773 (Kuimelis and Wagner, “Addressable proteinarrays,” Oct. 14, 1999), International publication WO 00/04389 (Wagneret al., “Arrays of protein-capture agents and methods of use thereof,”Jul. 27, 2000), International publication WO 00/56934 (Englert et al.,“Continuous porous matrix arrays,” Sep. 28, 2000).

In general, a sample containing the biomarkers is placed on the activesurface of a biochip for a sufficient time to allow binding. Then,unbound molecules are washed from the surface using a suitable eluant.In general, the more stringent the eluant, the more tightly the proteinsmust be bound to be retained after the wash. The retained proteinbiomarkers now can be detected by appropriate means.

Analytes captured on the surface of a protein biochip can be detected byany method known in the art. This includes, for example, massspectrometry, fluorescence, surface plasmon resonance, ellipsometry andatomic force microscopy. Mass spectrometry, and particularly SELDI massspectrometry, is a particularly useful method for detection of thebiomarkers of this invention.

Preferably, a laser desorption time-of-flight mass spectrometer is usedin embodiments of the invention. In laser desorption mass spectrometry,a substrate or a probe comprising markers is introduced into an inletsystem. The markers are desorbed and ionized into the gas phase by laserfrom the ionization source. The ions generated are collected by an ionoptic assembly, and then in a time-of-flight mass analyzer, ions areaccelerated through a short high voltage field and let drift into a highvacuum chamber. At the far end of the high vacuum chamber, theaccelerated ions strike a sensitive detector surface at a differenttime. Since the time-of-flight is a function of the mass of the ions,the elapsed time between ion formation and ion detector impact can beused to identify the presence or absence of markers of specific mass tocharge ratio.

Matrix-assisted laser desorption/ionization mass spectrometry, orMALDI-MS, is a method of mass spectrometry that involves the use of anenergy absorbing molecule, frequently called a matrix, for desorbingproteins intact from a probe surface. MALDI is described, for example,in U.S. Pat. No. 5,118,937 (Hillenkamp et al.) and U.S. Pat. No.5,045,694 (Beavis and Chait). In MALDI-MS the sample is typically mixedwith a matrix material and placed on the surface of an inert probe.Exemplary energy absorbing molecules include cinnamic acid derivatives,sinapinic acid (“SPA”), cyano hydroxy cinnamic acid (“CHCA”) anddihydroxybenzoic acid. Other suitable energy absorbing molecules areknown to those skilled in this art. The matrix dries, forming crystalsthat encapsulate the analyte molecules. Then the analyte molecules aredetected by laser desorption/ionization mass spectrometry. MALDI-MS isuseful for detecting the biomarkers of this invention if the complexityof a sample has been substantially reduced using the preparation methodsdescribed above.

Surface-enlianced laser desorption/ionization mass spectrometry, orSELDI-MS represents an improvement over MALDI for the fractionation anddetection of biomolecules, such as proteins, in complex mixtures. SELDIis a method of mass spectrometry in which biomolecules, such asproteins, are captured on the surface of a protein biochip using capturereagents that are bound there. Typically, non-bound molecules are washedfrom the probe surface before interrogation. SELDI is described, forexample, in: U.S. Pat. No. 5,719,060 (“Method and Apparatus forDesorption and Ionization of Analytes,” Hutchens and Yip, Feb. 17, 1998)U.S. Pat. No. 6,225,047 (“Use of Retentate Chromatography to GenerateDifference Maps,” Hutchens and Yip, May 1, 2001) and Weinberger et al.,“Time-of-flight mass spectrometry,” in Encyclopedia of AnalyticalChemistry, R. A. Meyers, ed., pp 11915-11918 John Wiley & SonsChichesher, 2000.

Markers on the substrate surface can be desorbed and ionized using gasphase ion spectrometry. Any suitable gas phase ion spectrometers can beused as long as it allows markers on the substrate to be resolved.Preferably, gas phase ion spectrometers allow quantitation of markers.

In one embodiment, a gas phase ion spectrometer is a mass spectrometer.In a typical mass spectrometer, a substrate or a probe comprisingmarkers on its surface is introduced into an inlet system of the massspectrometer. The markers are then desorbed by a desorption source suchas a laser, fast atom bombardment, high energy plasma, electrosprayionization, thermospray ionization, liquid secondary ion MS, fielddesorption, etc. The generated desorbed, volatilized species consist ofpreformed ions or neutrals which are ionized as a direct consequence ofthe desorption event. Generated ions are collected by an ion opticassembly, and then a mass analyzer disperses and analyzes the passingions. The ions exiting the mass analyzer are detected by a detector. Thedetector then translates information of the detected ions intomass-to-charge ratios. Detection of the presence of markers or othersubstances will typically involve detection of signal intensity. This,in turn, can reflect the quantity and character of markers bound to thesubstrate. Any of the components of a mass spectrometer (e.g., adesorption source, a mass analyzer, a detector, etc.) can be combinedwith other suitable components described herein or others known in theart in embodiments of the invention.

In another embodiment, an immunoassay can be used to detect and analyzemarkers in a sample. This method comprises: (a) providing an antibodythat specifically binds to a marker; (b) contacting a sample with theantibody; and (c) detecting the presence of a complex of the antibodybound to the marker in the sample.

To prepare an antibody that specifically binds to a marker, purifiedmarkers or their nucleic acid sequences can be used. Nucleic acid andamino acid sequences for markers can be obtained by furthercharacterization of these markers. The molecular weights of digestionfragments from each marker can be used to search the databases, such asSwissProt database, for sequences that will match the molecular weightsof digestion fragments generated by various enzymes. Using this method,the nucleic acid and amino acid sequences of other markers can beidentified if these markers are known proteins in the databases.

Alternatively, the proteins can be sequenced using protein laddersequencing. Protein ladders can be generated by, for example,fragmenting the molecules and subjecting fragments to enzymaticdigestion or other methods that sequentially remove a single amino acidfrom the end of the fragment. Methods of preparing protein ladders aredescribed, for example, in International Publication WO 93/24834 (Chaitet al.) and U.S. Pat. No. 5,792,664 (Chait et al.). The ladder is thenanalyzed by mass spectrometry. The difference in the masses of theladder fragments identify the amino acid removed from the end of themolecule.

If the markers are not known proteins in the databases, nucleic acid andamino acid sequences can be determined with knowledge of even a portionof the amino acid sequence of the marker. For example, degenerate probescan be made based on the N-terminal amino acid sequence of the marker.These probes can then be used to screen a genomic or cDNA librarycreated from a sample from which a marker was initially detected. Thepositive clones can be identified, amplified, and their recombinant DNAsequences can be subcloned using techniques which are well known. See,e.g., Current Protocols for Molecular Biology (Ausubel et al., GreenPublishing Assoc. and Wiley-Interscience 1989) and Molecular Cloning: ALaboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring HarborLaboratory, NY 2001).

Using the purified markers or their nucleic acid sequences, antibodiesthat specifically bind to a marker can be prepared using any suitablemethods known in the art. See, e.g., Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies. A Laboratory Manual(1988); Goding, Monoclonal Antibodies. Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude, but are not limited to, antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).

After the antibody is provided, a marker can be detected and/orquantified using any of suitable immunological binding assays known inthe art (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168). Useful assays include, for example, an enzyme immune assay(EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmuneassay (RIA), a Western blot assay, or a slot blot assay. These methodsare also described in, e.g., Methods in Cell Biology Antibodies in CellBiology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991); and Harlow & Lane, supra.

Generally, a sample obtained from a subject can be contacted with theantibody that specifically binds the marker. Optionally, the antibodycan be fixed to a solid support to facilitate washing and subsequentisolation of the complex, prior to contacting the antibody with asample. Examples of solid supports include glass or plastic in the formof, e.g., a microtiter plate, a stick, a bead, or a microbead.Antibodies can also be attached to a probe substrate or protein chiparray described above. The sample is preferably a biological fluidsample taken from a subject. Examples of biological fluid samplesinclude cerebrospinal fluid, blood, serum, plasma, neuronal cells,tissues, urine, tears, saliva etc. In a preferred embodiment, thebiological fluid comprises cerebrospinal fluid. The sample can bediluted with a suitable eluant before contacting the sample to theantibody.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed can be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.This detection reagent may be, e.g., a second antibody which is labeledwith a detectable label. Exemplary detectable labels include magneticbeads (e.g., DYNABEADS™), fluorescent dyes, radiolabels, enzymes (e.g.,horse radish peroxide, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic beads. Alternatively, the marker in the sample can bedetected using an indirect assay, wherein, for example, a second,labeled antibody is used to detect bound marker-specific antibody,and/or in a competition or inhibition assay wherein, for example, amonoclonal antibody which binds to a distinct epitope of the marker isincubated simultaneously with the mixture.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,marker, volume of solution, concentrations fragments and derivativesthereof. Usually the assays will be carried out at ambient temperature,although they can be conducted over a range of temperatures, such as 10°C. to 40° C.

Immunoassays can be used to determine presence or absence of a marker ina sample as well as the quantity of a marker in a sample. First, a testamount of a marker in a sample can be detected using the immunoassaymethods described above. If a marker is present in the sample, it willform an antibody-marker complex with an antibody that specifically bindsthe marker under suitable incubation conditions described above. Theamount of an antibody-marker complex can be determined by comparing to astandard. A standard can be, e.g., a known compound or another proteinknown to be present in a sample. As noted above, the test amount ofmarker need not be measured in absolute units, as long as the unit ofmeasurement can be compared to a control.

The methods for detecting these markers in a sample have manyapplications. For example, one or more markers can be measured to aid inthe diagnosis of spinal injury, brain injury, the degree of injury,neural injury due to neuronal disorders, alcohol and drug abuse, fetalinjury due to alcohol and/or drug abuse by pregnant mothers, etc. Inanother example, the methods for detection of the markers can be used tomonitor responses in a subject to treatment. In another example, themethods for detecting markers can be used to assay for and to identifycompounds that modulate expression of these markers in vivo or in vitro.

Data generated by desorption and detection of markers can be analyzedusing any suitable means. In one embodiment, data is analyzed with theuse of a programmable digital computer. The computer program generallycontains a readable medium that stores codes. Certain code can bedevoted to memory that includes the location of each feature on a probe,the identity of the adsorbent at that feature and the elution conditionsused to wash the adsorbent. The computer also contains code thatreceives as input, data on the strength of the signal at variousmolecular masses received from a particular addressable location on theprobe. This data can indicate the number of markers detected, includingthe strength of the signal generated by each marker.

Data analysis can include the steps of determining signal strength(e.g., height of peaks) of a marker detected and removing “outliers”(data deviating from a predetermined statistical distribution). Theobserved peaks can be normalized, a process whereby the height of eachpeak relative to some reference is calculated. For example, a referencecan be background noise generated by instrument and chemicals (e.g.,energy absorbing molecule) which is set as zero in the scale. Then thesignal strength detected for each marker or other biomolecules can bedisplayed in the form of relative intensities in the scale desired(e.g., 100). Alternatively, a standard (e.g., a CSF protein) may beadmitted with the sample so that a peak from the standard can be used asa reference to calculate relative intensities of the signals observedfor each marker or other markers detected.

The computer can transform the resulting data into various formats fordisplaying. In one format, referred to as “spectrum view or retentatemap,” a standard spectral view can be displayed, wherein the viewdepicts the quantity of marker reaching the detector at each particularmolecular weight. In another format, referred to as “peak map,” only thepeak height and mass information are retained from the spectrum view,yielding a cleaner image and enabling markers with nearly identicalmolecular weights to be more easily seen. In yet another format,referred to as “gel view,” each mass from the peak view can be convertedinto a grayscale image based on the height of each peak, resulting in anappearance similar to bands on electrophoretic gels. In yet anotherformat, referred to as “3-D overlays,” several spectra can be overlaidto study subtle changes in relative peak heights. In yet another format,referred to as “difference map view,” two or more spectra can becompared, conveniently highlighting unique markers and markers which areup- or down-regulated between samples. Marker profiles (spectra) fromany two samples may be compared visually. In yet another format,Spotfire Scatter Plot can be used, wherein markers that are detected areplotted as a dot in a plot, wherein one axis of the plot represents theapparent molecular mass of the markers detected and another axisrepresents the signal intensity of markers detected. For each sample,markers that are detected and the amount of markers present in thesample can be saved in a computer readable medium. This data can then becompared to a control (e.g., a profile or quantity of markers detectedin control, e.g., normal, healthy subjects in whom neural injury isundetectable).

Alternative Methods for Identification of Homologous ProteolyticBiomarkers

With respect to the cloning of allelic variants of the human proteolyticmarker genes and homologues from other species (e.g., mouse), isolatedproteolytic marker gene sequences may be labeled and used to screen acDNA library constructed from mRNA obtained from appropriate cells ortissues (e.g., brain tissues) derived from the organism (e.g., mouse) ofinterest. The hybridization conditions used should be of a lowerstringency when the cDNA library is derived from an organism differentfrom the type of organism from which the labeled sequence was derived.

Alternatively, the labeled fragment may be used to screen a genomiclibrary derived from the organism of interest, again, usingappropriately stringent conditions. Low stringency conditions are wellknown to those of skill in the art, and will vary predictably dependingon the specific organisms from which the library and the labeledsequences are derived. For guidance regarding such conditions see, forexample, Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Press, N.Y.; and Ausubel, et al.,1989, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y.

Further, an proteolytic marker gene allelic variant may be isolatedfrom, for example, human nucleic acid, by performing PCR using the panspecific probes. For example, the template for the reaction may be cDNAobtained by reverse transcription of mRNA prepared from, for example,human or non-human cell lines or tissue known or suspected to express aproteolytic marker gene or allelic variant thereof. Preferably, theallelic variant will be isolated from an individual who has a neuronaldisorder.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express the proteolytic marker gene, such as,for example, brain tissue samples obtained through biopsy orpost-mortem). A reverse transcription reaction may be performed on theRNA using an oligonucleotide primer specific for the most 5′ end of theamplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” with guanines using astandard terminal transferase reaction, the hybrid may be digested withRNAse H, and second strand synthesis may then be primed with a poly-Cprimer. Thus, cDNA sequences upstream of the amplified fragment mayeasily be isolated. For a review of cloning strategies that may be used,see e.g., Sambrook et al., 1989, infra.

Another preferred method includes SAGE. Serial Analysis of GeneExpression (SAGE), is based on the identification of andcharacterization of partial, defined sequences of transcriptscorresponding to gene segments. These defined transcript sequence “tags”are markers for genes which are expressed in a cell, a tissue, or anextract, for example.

SAGE is based on several principles. First, a short nucleotide sequencetag (9 to 10 bp) contains sufficient information content to uniquelyidentify a transcript provided it is isolated from a defined positionwithin the transcript. For example, a sequence as short as 9 bp candistinguish about 262,144 transcripts given a random nucleotidedistribution at the tag site, whereas estimates suggest that the humangenome encodes about 80,000 to 200,000 transcripts (Fields, et al.,Nature Genetics, 7:345 1994). The size of the tag can be shorter forlower eukaryotes or prokaryotes, for example, where the number oftranscripts encoded by the genome is lower. For example, a tag as shortas 6-7 bp may be sufficient for distinguishing transcripts in yeast.

Second, random dimerization of tags allows a procedure for reducing bias(caused by amplification and/or cloning). Third, concatenation of theseshort sequence tags allows the efficient analysis of transcripts in aserial manner by sequencing multiple tags within a single vector orclone. As with serial communication by computers, wherein information istransmitted as a continuous string of data, serial analysis of thesequence tags requires a means to establish the register and boundariesof each tag. The concept of deriving a defined tag from a sequence inaccordance with the present invention is useful in matching tags ofsamples to a sequence database. In the preferred embodiment, a computermethod is used to match a sample sequence with known sequences.

The tags used herein, uniquely identify genes. This is due to theirlength, and their specific location (3′) in a gene from which they aredrawn. The full length genes can be identified by matching the tag to agene data base member, or by using the tag sequences as probes tophysically isolate previously unidentified genes from cDNA libraries.The methods by which genes are isolated from libraries using DNA probesare well known in the art. See, for example, Veculescu et al., Science270: 484 (1995), and Sambrook et al. (1989), MOLECULAR CLONING: ALABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). Once a gene or transcript has been identified, either bymatching to a data base entry, or by physically hybridizing to a cDNAmolecule, the position of the hybridizing or matching region in thetranscript can be determined. If the tag sequence is not in the 3′ end,immediately adjacent to the restriction enzyme used to generate the SAGEtags, then a spurious match may have been made. Confirmation of theidentity of a SAGE tag can be made by comparing transcription levels ofthe tag to that of the identified gene in certain cell types. Analysisof gene expression is not limited to the above method but can includeany method known in the art. All of these principles may be appliedindependently, in combination, or in combination with other knownmethods of sequence identification.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

In a preferred embodiment, Expressed Sequenced Tags (ESTs), can also beused to identify nucleic acid molecules which are over expressed in aneuronal cell. ESTs from a variety of databases can be indentified. Forexample, preferred databases include, for example, Online MendelianInheritance in Man (OMIM), the Cancer Genome Anatomy Project (CGAP),GenBank, EMBL, PIR, SWISS-PROT, and the like. OMIM, which is a databaseof genetic mutations associated with disease, was developed, in part,for the National Center for Biotechnology Information (NCBI). OMIM canbe accessed through the world wide web of the Internet, at, for example,ncbi.nlm.nih.gov/Omim/. CGAP, which is an interdisciplinary program toestablish the information and technological tools required to decipherthe molecular anatomy of a cancer cell. CGAP can be accessed through theworld wide web of the Internet, at, for example,ncbi.nlm.nih.gov/ncicgap/. Some of these databases may contain completeor partial nucleotide sequences. In addition, alternative transcriptforms can also be selected from private genetic databases.Alternatively, nucleic acid molecules can be selected from availablepublications or can be determined especially for use in connection withthe present invention.

Alternative transcript forms can be generated from individual ESTs whichare within each of the databases by computer software which generatescontiguous sequences. In another embodiment of the present invention,the nucleotide sequence of the nucleic acid molecule is determined byassembling a plurality of overlapping ESTs. The EST database (dbEST),which is known and available to those skilled in the art, comprisesapproximately one million different human mRNA sequences comprising fromabout 500 to 1000 nucleotides, and various numbers of ESTs from a numberof different organisms. dbEST can be accessed through the world wide webof the Internet, at, for example, ncbi.nlm.nih.gov/dbEST/index.html.These sequences are derived from a cloning strategy that uses cDNAexpression clones for genome sequencing. ESTs have applications in thediscovery of new genes, mapping of genomes, and identification of codingregions in genomic sequences. Another important feature of EST sequenceinformation that is becoming rapidly available is tissue-specific geneexpression data. This can be extremely useful in targeting selectivegene(s) for therapeutic intervention. Since EST sequences are relativelyshort, they must be assembled in order to provide a complete sequence.Because every available clone is sequenced, it results in a number ofoverlapping regions being reported in the database. The end result isthe elicitation of alternative transcript forms from, for example,immune cells and neuronal cells.

Assembly of overlapping ESTs extended along both the 5′ and 3′directions results in a full-length “virtual transcript.” The resultantvirtual transcript may represent an already characterized nucleic acidor may be a novel nucleic acid with no known biological function. TheInstitute for Genomic Research (TIGR) Human Genome Index (HGI) database,which is known and available to those skilled in the art, contains alist of human transcripts. TIGR can be accessed through the world wideweb of the Internet, at, for example, tigr.org. Transcripts can begenerated in this manner using TIGR-Assembler, an engine to buildvirtual transcripts and which is known and available to those skilled inthe art. TIGR-Assembler is a tool for assembling large sets ofoverlapping sequence data such as ESTs, BACs, or small genomes, and canbe used to assemble eukaryotic or prokaryotic sequences. TIGR-Assembleris described in, for example, Sutton, et al., Genome Science & Tech.,1995, 1, 9-19, which is incorporated herein by reference in itsentirety, and can be accessed through the file transfer program of theInternet, at, for example, tigr.org/pub/software/TIGR. assembler. Inaddition, GLAXO-MRC, which is known and available to those skilled inthe art, is another protocol for constructing virtual transcripts. PHRAPis used for sequence assembly within Find Neighbors and Assemble ESTBlast. PHRAP can be accessed through the world wide web of the Internet,at, for example, chimera.biotech.washington.edu/uwgc/tools/phrap.htm.Identification of ESTs and generation of contiguous ESTs to form fulllength RNA molecules is described in detail in U.S. application Ser. No.09/076,440, which is incorporated herein by reference in its entirety.

As mentioned above, the proteolytic marker gene sequences may be used toisolate mutant proteolytic marker gene alleles, preferably from a humansubject. Such mutant alleles may be isolated from individuals eitherknown or proposed to have a genotype that contributes to the symptoms ofa neuronal disorder such as Alzheimer's or Parkinson's disease.

A cDNA of a mutant allelic variant of the proteolytic marker gene may beisolated, for example, by using PCR, a technique that is well known tothose of skill in the art. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known or suspected to be expressed in an individualputatively carrying the mutant proteolytic marker allele, and byextending the new strand with reverse transcriptase. The second strandof the cDNA is then synthesized using an oligonucleotide that hybridizesspecifically to the 5′ end of the normal gene. Using these two primers,the product is then amplified via PCR, cloned into a suitable vector,and subjected to DNA sequence analysis through methods well known tothose of skill in the art. By comparing the DNA sequence of the mutantproteolytic marker allele to that of the normal proteolytic markerallele, the mutation(s) responsible for the loss or alteration offunction of the mutant proteolytic marker gene product can beascertained.

Genomic DNA isolated from lymphocytes or other immune cells of normaland affected individuals can also be used as PCR template. PCR productsfrom normal and affected individuals are compared, either by singlestrand conformational polymorphism (SSCP) mutation detection techniquesand/or by sequencing.

In another embodiment of the invention, the above nucleic acid sequencesencoding proteolytic markers may be used to generate hybridizationprobes useful in mapping the naturally-occurring genomic sequence, aswell as to detect in an individual, or group of individuals, allelicvariants of genes that are present in individuals suffering from orsusceptible to neural defects or diseases. The sequences may be mappedto a particular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial PI constructions, or single chromosomecDNA libraries (see, e.g., Harrington et al., 1997, Nat. Genet. 15:345-355; Price, 1993, Blood Rev. 7: 127-134; and Trask, 1991, TrendsGenet. 7: 149-154).

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data (see, e.g.,Heinz-Ulrich et al., 1995, in Meyers, supra, pp. 965-968). Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. The nucleotidesequences of the invention may be used to detect differences in genesequences among resistant, susceptible, or allelic variants inindividuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for genes of the invention using positionalcloning or other gene discovery techniques. Once the genes have beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation (see, e.g., Gatti et al., 1988, Nature 336:577-580). Otherexamples of particular genomic regions include, but not limited to,leukocyte receptor cluster to 19q13.3-13.4 The nucleotide sequence ofthe subject invention may also be used to detect differences in thechromosomal location due to translocation, inversion, etc., amongnormal, or affected individuals.

The genes identified from individuals are amplified by PCR and sequencedby methods well known in the art. These nucleic acid sequences are thenused in the assays described in the examples and materials and methodsto correlate the sequence of the genes identified, with, for example,the percentage of individuals suffering from Alzheimer's disease ascompared to normal individuals. As more gene sequences and their aminoacid sequences are identified, allows for a correlation between theexpression of proteolytic markers in cells of the nervous system,including the brain, and individuals predisposed to a neural disease ordefect.

In yet another aspect, variants of the nucleic acid molecules asidentified in immune cells from individuals of different haplotypesand/or suffering from or susceptible to neural defects can be used todetect allelic variations of immune related molecules in neural cells.An “allele” or “variant” is an alternative form of a gene. Of particularutility in the invention are variants of the genes encoding anypotential immune related molecule in the nervous system identified bythe methods of this invention. Variants may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orin polypeptides whose structure or function may or may not be altered.Any given natural or recombinant gene may have none, one, or manyallelic forms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

To further identify variant nucleic acid molecules which can detect, forexample, early stage Alzheimer's, nucleic acid molecules can be groupedinto sets depending on the homology, for example. The members of a setof nucleic acid molecules are compared. Preferably, the set of nucleicacid molecules is a set of alternative transcript forms of nucleic acid.Preferably, the members of the set of alternative transcript forms ofnucleic acids include at least one member which is associated, or whoseencoded protein is associated, with a disease state or biologicalcondition. For example, a set of proteolytic marker molecules from braincells and neural cells from normal and a diseased individual arecompared. At least one of the members of the set of nucleic acidmolecule alternative transcript forms can be associated with forexample, Alzheimer's or any other neural defect, as described above.Thus, comparison of the members of the set of nucleic acid moleculesresults in the identification of at least one alternative transcriptform of nucleic acid molecule which is associated, or whose encodedprotein is associated, with a disease state or biological condition. Ina preferred embodiment of the invention, the members of the set ofnucleic acid molecules are from a common gene. In another embodiment ofthe invention, the members of the set of nucleic acid molecules are froma plurality of genes. In another embodiment of the invention, themembers of the set of nucleic acid molecules are from differenttaxonomic species. Nucleotide sequences of a plurality of nucleic acidsfrom different taxonomic species can be identified by performing asequence similarity search, an ortholog search, or both, such searchesbeing known to persons of ordinary skill in the art.

Sequence similarity searches can be performed manually or by usingseveral available computer programs known to those skilled in the art.Preferably, Blast and Smith-Waterman algorithms, which are available andknown to those skilled in the art, and the like can be used. Blast isNCBI's sequence similarity search tool designed to support analysis ofnucleotide and protein sequence databases. Blast can be accessed throughthe world wide web of the Internet, at, for example,ncbi.nlm.nih.gov/BLAST/. The GCG Package provides a local version ofBlast that can be used either with public domain databases or with anylocally available searchable database. GCG Package v9.0 is acommercially available software package that contains over 100interrelated software programs that enables analysis of sequences byediting, mapping, comparing and aligning them. Other programs includedin the GCG Package include, for example, programs which facilitate RNAsecondary structure predictions, nucleic acid fragment assembly, andevolutionary analysis. In addition, the most prominent genetic databases(GenBank, EMBL, PIR, and SWISS-PROT) are distributed along with the GCGPackage and are fully accessible with the database searching andmanipulation programs. GCG can be accessed through the Internet at, forexample, http://www.gcg.com/. Fetch is a tool available in GCG that canget annotated GenBank records based on accession numbers and is similarto Entrez. Another sequence similarity search can be performed withGeneWorld and GeneThesaurus from Pangea. GeneWorld 2.5 is an automated,flexible, high-throughput application for analysis of polynucleotide andprotein sequences. GeneWorld allows for automatic analysis andannotations of sequences. Like GCG, GeneWorld incorporates several toolsfor homology searching, gene finding, multiple sequence alignment,secondary structure prediction, and motif identification. GeneThesaurus1.0™ is a sequence and annotation data subscription service providinginformation from multiple sources, providing a relational data model forpublic and local data.

Another alternative sequence similarity search can be performed, forexample, by BlastParse. BlastParse is a PERL script running on a UNIXplatform that automates the strategy described above. BlastParse takes alist of target accession numbers of interest and parses all the GenBankfields into “tab-delimited” text that can then be saved in a “relationaldatabase” format for easier search and analysis, which providesflexibility. The end result is a series of completely parsed GenBankrecords that can be easily sorted, filtered, and queried against, aswell as an annotations-relational database.

Preferably, the plurality of nucleic acids from different taxonomicspecies which have homology to the target nucleic acid, as describedabove in the sequence similarity search, are further delineated so as tofind orthologs of the target nucleic acid therein. An ortholog is a termdefined in gene classification to refer to two genes in widely divergentorganisms that have sequence similarity, and perform similar functionswithin the context of the organism. In contrast, paralogs are geneswithin a species that occur due to gene duplication, but have evolvednew functions, and are also referred to as isotypes. Optionally, paralogsearches can also be performed. By performing an ortholog search, anexhaustive list of homologous sequences from as diverse organisms aspossible is obtained. Subsequently, these sequences are analyzed toselect the best representative sequence that fits the criteria for beingan ortholog. An ortholog search can be performed by programs availableto those skilled in the art including, for example, Compare. Preferably,an ortholog search is performed with access to complete and parsedGenBank annotations for each of the sequences. Currently, the recordsobtained from GenBank are “flat-files”, and are not ideally suited forautomated analysis. Preferably, the ortholog search is performed using aQ-Compare program. Preferred steps of the Q-Compare protocol aredescribed in the flowchart set forth in U.S. Pat. No. 6,221,587,incorporated herein by reference.

Preferably, interspecies sequence comparison is performed using Compare,which is available and known to those skilled in the art. Compare is aGCG tool that allows pair-wise comparisons of sequences using awindow/stringency criterion. Compare produces an output file comprisingpoints where matches of specified quality are found. These can beplotted with another GCG tool, DotPlot.

The polynucleotides of this invention can be isolated using thetechnique described in the experimental section or replicated using PCR.The PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065, and 4,683,202 and described in PCR: The PolymeraseChain Reaction (Mullis et al. eds, Birkhauser Press, Boston (1994)) orMacPherson et al. (1991) and (1994), supra, and references citedtherein. Alternatively, one of skill in the art can use the sequencesprovided herein and a commercial DNA synthesizer to replicate the DNA.Accordingly, this invention also provides a process for obtaining thepolynucleotides of this invention by providing the linear sequence ofthe polynucleotide, nucleotides, appropriate primer molecules, chemicalssuch as enzymes and instructions for their replication and chemicallyreplicating or linking the nucleotides in the proper orientation toobtain the polynucleotides. In a separate embodiment, thesepolynucleotides are further isolated. Still further, one of skill in theart can insert the polynucleotide into a suitable replication vector andinsert the vector into a suitable host cell (procaryotic or eucaryotic)for replication and amplification. The DNA so amplified can be isolatedfrom the cell by methods well known to those of skill in the art. Aprocess for obtaining polynucleotides by this method is further providedherein as well as the polynucleotides so obtained.

In an embodiment of the invention the presence of the one or moreproteolytic marker nucleic acid molecules, isolated from a cell, iscorrelated to neuronal cell sample of a normal subject and one sufferingfrom or susceptible to a neural disorder. The sample is preferablyobtained from a mammal suspected of having a nerve or brain celldisorder. Preferably, a nucleic acid molecule that is indicative of aproteolytic marker molecule and detected in a neural cell comprises asequence having at least about 50% sequence identity to any one of SEQID NO's: 1-148, more preferably a nucleic acid molecule that isindicative of a proteolytic marker molecule and detected in a neuralcell comprises a sequence having at least about 70% sequence identity toany one of SEQ ID NO's: 1-148, more preferably a nucleic acid moleculethat is indicative of a proteolytic marker molecule and detected in aneural cell comprises a sequence having at least about 80% sequenceidentity to any one of SEQ ID NO's: 1-148, more preferably a nucleicacid molecule that is indicative of a proteolytic marker molecule anddetected in a neural cell comprises a sequence having at least about90%, 95%, 96%, 97%, 95%, 99% or 99.9% sequence identity to any one ofSEQ ID NO's: 1-148.

Percent identity and similarity between two sequences (nucleic acid orpolypeptide) can be determined using a mathematical algorithm (see,e.g., Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991).

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps are introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap which need to beintroduced for optimal alignment of the two sequences. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions, respectively, are then compared. When a positionin the first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position (as used herein amino acidor nucleic acid “identity” is equivalent to amino acid or nucleic acid“homology”).

A “comparison window” refers to a segment of any one of the number ofcontiguous positions selected from the group consisting of from about 25to about 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art.

For example, the percent identity between two amino acid sequences canbe determined using the Needleman and Wunsch algorithm (J. Mol. Biol.(48): 444-453, 1970) which is part of the GAP program in the GCGsoftware package (available at http://www.gcg.com), by the localhomology algorithm of Smith & Waterman (Adv. Appl. Math. 2: 482, 1981),by the search for similarity methods of Pearson & Lipman (Proc. Natl.Acad. Sci. USA 85: 2444, 1988) and Altschul, et al. (Nucleic Acids Res.25(17): 3389-3402, 1997), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and BLAST in the Wisconsin GeneticsSoftware Package (available from, Genetics Computer Group, 575 ScienceDr., Madison, Wis.), or by manual alignment and visual inspection (see,e.g., Ausubel et al., supra). Gap parameters can be modified to suit auser's needs. For example, when employing the GCG software package, aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6 can be used. Exemplary gap weightsusing a Blossom 62 matrix or a PAM250 matrix, are 16, 14, 12, 10, 8, 6,or 4, while exemplary length weights are 1, 2, 3, 4, 5, or 6. The GCGsoftware package can be used to determine percent identity betweennucleic acid sequences. The percent identity between two amino acid ornucleotide sequences also can be determined using the algorithm of E.Myers and W. Miller (CABIOS 4: 11-17, 1989) which has been incorporatedinto the ALIGN program (version 2.0), using a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as query sequences to perform a search against sequencedatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (J. Mol. Biol. 215: 403-10,1990). BLAST nucleotide searches can be performed with the NBLASTprogram, with exemplary scores=100, and wordlengths=12 to obtainnucleotide sequences homologous to or with sufficient percent identityto the nucleic acid molecules of the invention. BLAST protein searchescan be performed with the XBLAST program, with exemplary scores=50 andwordlengths=3 to obtain amino acid sequences sufficiently homologous toor with sufficient % identity to the proteins of the invention. Toobtain gapped alignments for comparison purposes, gapped BLAST can beused as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402, 1997). When using BLAST and gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

The invention also comprises polypeptides, in neural cells,corresponding to a nucleic acid molecule product which comprisesconservative substitutions that are phenotypically silent. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Guidanceconcerning amino acid changes which are likely to be phenotypicallysilent may be found in Bowie et al., Science 247: 1306-1310, 1990, forexample. Conservative substitution tables providing functionally similaramino acids are well known in the art (see, e.g., Henikoff and Henikoff,Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992) and in the tablebelow.

Conservative Amino Acid Substitutions Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

As used herein, the term “fragment or segment”, as applied to a nucleicacid sequence, gene or polypeptide, will ordinarily be at least about 5contiguous nucleic acid bases (for nucleic acid sequence or gene) oramino acids (for polypeptides), typically at least about 10 contiguousnucleic acid bases or amino acids, more typically at least about 20contiguous nucleic acid bases or amino acids, usually at least about 30contiguous nucleic acid bases or amino acids, preferably at least about40 contiguous nucleic acid bases or amino acids, more preferably atleast about 50 contiguous nucleic acid bases or amino acids, and evenmore preferably at least about 60 to 80 or more contiguous nucleic acidbases or amino acids in length. “Overlapping fragments” as used herein,refer to contiguous nucleic acid or peptide fragments which begin at theamino terminal end of a nucleic acid or protein and end at the carboxyterminal end of the nucleic acid or protein. Each nucleic acid orpeptide fragment has at least about one contiguous nucleic acid or aminoacid position in common with the next nucleic acid or peptide fragment,more preferably at least about three contiguous nucleic acid bases oramino acid positions in common, most preferably at least about tencontiguous nucleic acid bases amino acid positions in common.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least 20nucleotides, more generally at least 23 nucleotides, ordinarily at least26 nucleotides, more ordinarily at least 29 nucleotides, often at least32 nucleotides, more often at least 35 nucleotides, typically at least38 nucleotides, more typically at least 41 nucleotides, usually at least44 nucleotides, more usually at least 47 nucleotides, preferably atleast 50 nucleotides, more preferably at least 53 nucleotides, and inparticularly preferred embodiments will be at least 56 or morenucleotides. Additional preferred embodiments will include lengths inexcess of those numbers, e.g., 63, 72, 87, 96, 105, 117, etc. Saidfragments may have termini at any pairs of locations, but especially atboundaries between structural domains, e.g., membrane spanning portions.

Homologous nucleic acid sequences, when compared, exhibit significantsequence identity or similarity. The standards for homology in nucleicacids are either measures for homology generally used in the art bysequence comparison or based upon hybridization conditions. Thehybridization conditions are described in greater detail below.

As used herein, “substantial homology” in the nucleic acid sequencecomparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least 56%, moregenerally at least 59%, ordinarily at least 62%, more ordinarily atleast 65%, often at least 68%, more often at least 71%, typically atleast 74%, more typically at least 77%, usually at least 80%, moreusually at least about 85%, preferably at least about 90%, morepreferably at least about 95 to 98% or more, and in particularembodiments, as high at about 99% or more of the nucleotides.Alternatively, substantial homology exists when the segments willhybridize under selective hybridization conditions, to a strand, or itscomplement, typically using a fragment derived from, for example, neuralor brain cells. Typically, selective hybridization will occur when thereis at least about 55% homology over a stretch of at least about 14nucleotides, preferably at least about 65%, more preferably at leastabout 75%, and most preferably at least about 90%. See, Kanehisa (1984)Nucl. Acids Res. 12:203-213. The length of homology comparison, asdescribed, may be over longer stretches, and in certain embodiments willbe over a stretch of at least about 17 nucleotides, usually at leastabout 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about40 nucleotides, preferably at least about 50 nucleotides, and morepreferably at least about 75 to 100 or more nucleotides. The endpointsof the segments may be at many different pair combinations. Chromosomalsynteny may be used to further distinguish between homologous genes whenthere is sufficient evolutionary conservation between the genomes thatare being compared, e.g. between mammalian species. A “syntenic homolog”has both sequence identity to the reference gene, and has thecorresponding chromosomal location in relation to closely linked genes.Syntenic homologs have a high probability of sharing spatial andtemporal localization of gene expression, and of encoding proteins thatfill equivalent biological roles.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., more usually in excessof about 37° C., typically in excess of about 45° C., more typically inexcess of about 55° C., preferably in excess of about 65° C., and morepreferably in excess of about 70° C. Stringent salt conditions willordinarily be less than about 1000 mM, usually less than about 500 mM,more usually less than about 400 mM, typically less than about 300 mM,preferably less than about 200 mM, and more preferably less than about150 mM. However, the combination of parameters is much more importantthan the measure of any single parameter. See, e.g., Wetmur and Davidson(1968) J. Mol. Biol. 31:349-370.

Diagnosis of Neural Injury

In another aspect, the invention provides methods for aiding a humanneural injury and/or neural disorder diagnosis using one or moremarkers. For example, proteolytic enzyme biomarkers with a specificactivity of about 1 μg to about 500 μg per 1 mg of substrate protein perbeing proteolyzed in injured or stressed organs (in vivo) within minutesto days after or in vitro using purified protease-substrateprotein/protein mixture ratio of 1/10,000 to 1/20 at a time point withinminutes to hours, for proteins identified in Table 1, peptides,fragments or derivatives thereof and or those identified by SEQ IDNO's.: 1-148. These markers can be used singularly or in combinationwith other proteolytic enzyme markers or a combination of proteolyticenzyme biomarkers and neural peptides. The markers are differentiallypresent in samples of a human patient, for example a TBI patient, and anormal subject in whom neural injury is undetectable. For example, someof the markers are expressed at an elevated level and/or are present ata higher frequency in human patients with neural injury and/or neuronaldisorders than in normal subjects. Therefore, detection of one or moreof these markers in a person would provide useful information regardingthe probability that the person may have neural injury and/or neuronaldisorder.

Nervous system diseases, neuronal disorders, and/or conditions, whichcan be treated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases, disorders, and/or conditions which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated, prevented,and/or diagnosed in a patient (including human and non-human mammalianpatients) according to the invention, include but are not limited to,the following lesions of either the central (including spinal cord,brain) or peripheral nervous systems: (1) ischemic lesions, in which alack of oxygen in a portion of the nervous system results in neuronalinjury or death, including cerebral infarction or ischemia, or spinalcord infarction or ischemia; (2) traumatic lesions, including lesionscaused by physical injury or associated with surgery, for example,lesions which sever a portion of the nervous system, or compressioninjuries; (3) malignant lesions, in which a portion of the nervoussystem is destroyed or injured by malignant tissue which is either anervous system associated malignancy or a malignancy derived fromnon-nervous system tissue; (4) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, forexample, by an abscess or associated with infection by humanimmunodeficiency virus, herpes zoster, or herpes simplex virus or withLyme disease, tuberculosis, syphilis; (5) degenerative lesions, in whicha portion of the nervous system is destroyed or injured as a result of adegenerative process including but not limited to degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases, disorders, and/or conditions, in which aportion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

Accordingly, embodiments of the invention include methods for aidinghuman neural injury and/or neuronal disorders, wherein the methodcomprises: (a) detecting at least one proteolytic enzyme biomarker in asample, and (b) correlating the detection of the marker or markers witha probable diagnosis of human neural injury and/or neuronal disorder ororgan injury such as the heart. The correlation may take into accountthe amount of the marker or markers in the sample compared to a controlamount of the marker or markers (up or down regulation of the marker ormarkers) (e.g., in normal subjects in whom human neural injury isundetectable). The correlation may take into account the presence orabsence of the markers in a test sample and the frequency of detectionof the same markers in a control. The correlation may take into accountboth of such factors to facilitate determination of whether a subjecthas neural injury, the degree of severity of the neural injury, andsubcellular location of the injury, or not.

Any suitable samples can be obtained from a subject to detect markers.Preferably, a sample is a cerebrospinal fluid sample from the subject.If desired, the sample can be prepared as described above to enhancedetectability of the markers. For example, to increase the detectabilityof markers, a blood serum sample from the subject can be preferablyfractionated by, e.g., Cibacron blue agarose chromatography and singlestranded DNA affinity chromatography, anion exchange chromatographyfragments and derivatives thereof. Sample preparations, such aspre-fractionation protocols, is optional and may not be necessary toenhance detectability of markers depending on the methods of detectionused. For example, sample preparation may be unnecessary if antibodiesthat specifically bind markers are used to detect the presence ofmarkers in a sample.

Any suitable method can be used to detect a marker or markers in asample. For example, an fluorogenic assays or gas phase ion spectrometrycan be used as described above. Using these methods, one or more markerscan be detected. Preferably, a sample is tested for the presence of aplurality of markers. Detecting the presence of a plurality of markers,rather than a single marker alone, would provide more information forthe diagnostician. Specifically, the detection of a plurality of markersin a sample would increase the percentage of true positive and truenegative diagnoses and would decrease the percentage of false positiveor false negative diagnoses.

The detection of the marker or markers is then correlated with aprobable diagnosis of neural injury and/or neuronal disorders. In someembodiments, the detection of the mere presence or absence of a marker,without quantifying the amount of marker, is useful and can becorrelated with a probable diagnosis of neural injury and/or neuronaldisorders.

In other embodiments, the detection of markers can involve quantifyingthe markers to correlate the detection of markers with a probablediagnosis of neural injury, degree of severity of neural injury,diagnosis of neural disorders fragments and derivatives thereof. Thus,if the amount of the markers detected in a subject being tested ishigher compared to a control amount, then the subject being tested has ahigher probability of having such injuries and/or neural disorders.

Similarly, in another embodiment, the detection of markers can furtherinvolve quantifying the markers to correlate the detection of markerswith a probable diagnosis of neural injury, degree of severity of neuralinjury, diagnosis of neural disorders, wherein the markers are presentin lower quantities in CSF or blood serum samples from patients than inblood serum samples of normal subjects. Thus, if the amount of themarkers detected in a subject being tested is lower compared to acontrol amount, then the subject being tested has a higher probabilityof having neural injury, organ injury and/or neural disorder.

When the markers are quantified, it can be compared to a control. Acontrol can be, e.g., the average or median amount of marker present incomparable samples of normal subjects in whom neural injury and/orneural disorders, is undetectable. The control amount is measured underthe same or substantially similar experimental conditions as inmeasuring the test amount. For example, if a test sample is obtainedfrom a subject's cerebrospinal fluid and/or blood serum sample and amarker is detected using a particular probe, then a control amount ofthe marker is preferably determined from a serum sample of a patientusing the same probe. It is preferred that the control amount of markeris determined based upon a significant number of samples from normalsubjects who do not have neural injury and/or neuronal disorders so thatit reflects variations of the marker amounts in that population.

Data generated by mass spectrometry can then be analyzed by a computersoftware. The software can comprise code that converts signal from themass spectrometer into computer readable form. The software also caninclude code that applies an algorithm to the analysis of the signal todetermine whether the signal represents a “peak” in the signalcorresponding to a marker of this invention, or other useful markers.The software also can include code that executes an algorithm thatcompares signal from a test sample to a typical signal characteristic of“normal” and human neural injury and determines the closeness of fitbetween the two signals. The software also can include code indicatingwhich the test sample is closest to, thereby providing a probablediagnosis.

In a preferred embodiment, the biomarkers are detected in a wide rangeof species, such as for example, mammals, bird, fish, reptiles. Forexample, synaptotagmin-1 is found in many species and is highlyconserved. Below is an example of synaptotagmin-1 taxonomic data.

Tax BLAST Report Query gi|35086 Synaptotagmin-1 (Synaptotagmin I)Taxonomy Report Bilateria 200 hits 23 orgs [root; cellular organisms;Eukaryota; Fungi/Metazoa group; Metazoa; Eumetazoa] Coelomata 195 hits22 orgs Chordata 170 hits 18 orgs [Deuterostomia] Gnathostomata 163 hits14 orgs [Craniata; Vertebrata] Euteleostomi 163 hits 13 orgs[Teleostomi] Tetrapoda 142 hits 11 orgs [Sarcopterygii] Amniota 141 hits10 orgs Estherio 134 hits 3 orgs [Mammalia; Theria] Laurasiatheria 24hits 2 orgs Bos taurus 11 hits 1 orgs [Cetartiodactyla; Ruminantia;Pecora; Bovidae; Bovinae; Bos] Canis familiaris 13 hits 1 orgs[Carnivora; Fissopedia; Canidae; Canis] Euarchontoglires 110 hits 7 orgsCatarrhini 33 hits 4 orgs [Primates; Simiiformes] Hominidae 36 hits 3orgs [Hominoidea] Pongo pygmeus 4 hits 1 orgs [Pongo] Homo/Pan/Gorillagroup 30 hits 2 orgs Homo sapiens 20 hits 1 orgs [Homo] Pan troglodytes10 hits 1 orgs [Pan] Macaca fascicularis 7 hits 1 orgs[Cercopithecoidea; Cercopithecidae; Cercopitheoinae; Macaca] Murinae 71hits 3 orgs [Glires; Rodentia; Sciurognathi; Muridae] Rattus 43 hits 2orgs Rattus rattus 31 hits 1 orgs Rattus norvegicus 40 hits 1 orgs Musmusculus 26 hits 1 orgs [Mus] Gallus gallus 7 hits 1 orgs [Sauropsida;Sauria; Archosauria; Aves; Neognathae; Galliforme; Phasianidae;Phasianinae; Gaullus] Xenopus laevis 1 hits 1 orgs [Amphibia; Batrachia;Anura; Mesobatrachia; Pipoidea; Pipidae; Xenopodinae; Xenopus; Xenopus]Clupeocephala 23 hits 2 orgs [Actinopterygii; Actinopteri; Neopterygii;Teleostei; Elopocephala] Danio rerio 11 hits 1 orgs [Otocephala;Ostariophysi; Otophysi; Cypriniphysi; Cypriniformes; Cyprinoidea;Cyprinidae; Rasborinae; Danio] Tetraodon nigroviridis 12 hits 1 orgs[Euteleostei; Neognathi; Neoteleostei; Eurypterygii; Ctenosquamata;Acanthomorpha; Euacanthomorpha; Holacanthopt] Discopyge ommata 4 hits 1orgs [Chondrichthyes; Elasmobranchii; Neoselachii; Squalea;Hypnosqualea; Pristiorajea; Batoidea; Torpediniformes] Halocynthiaroretzi 1 hits 1 orgs [Urochordata; Ascidiacea; Stolidobranchia;Pyuridae; Halocynthia] Protostomia 18 hits 7 orgs Mollusca 3 hits 3 orgsGastropoda 3 hits 2 orgs Lymnaea stagnalis 1 hits 1 orgs [Pulmonata;Basommatophora; Lymnaeoidea; Lymnaeidae; Lymnaea] Aplysia californica 2hits 1 orgs [Orthogastropoda; Apogastropoda; Heterobranchia; Euthyneura;Opisthobranchia; Anaspidea; Aplysioidea; Aplysiidae] Loligo pealei 2hits 1 orgs [Cephalopoda; Coleoidea; Neocoleoidea; Decapodiformes;Loliginidae; Loligo] Endopterygota 13 hits 4 orgs [Panarthropoda;Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia;Pterygota] Apis mellifera 2 hits 1 orgs [Hymenoptera; Aculeata; Apoidea;Apidae; Apinae; Apini; Apis] Manduca sexta 2 hits 1 orgs[Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura;Ditrysia; Obtectomera; Bombycoidea; Sphin] Sophophora 3 hits 2 orgs[Diptera; Brachycera; Muscomorpha; Schizophora; Acalyptratae;Ephydroidea; Drosophilidae; Droso] Drosophila melanogaster 2 hits 1 orgs[melanogaster group; melanogaster subgroup] Drosophila pseudoobscura 1hits 1 orgs [obscura group; pseudoobscura subgroup] Dugesia japonica 1hits 1 orgs [Acoelomata; Platyhelminthes; Turbellaria; Seriata;Tricladida; Paludicola; Dugesiidae; Dugesia] Caenorhabditis 11 hits 2orgs [Pseudocoelomata; Nematoda; Chromadorea; Rhabditida; Rhabditoidea;Rhabditidae; Peloderinae] Caenorhabditis elegans 7 hits 1 orgsCaenorhabditis briggsae 4 hits 1 orgs

See WorldWide Website: ncbi.nlm.nih.gov/sutils/iqtax.cgi

Production of Antibodies to Detect Cleavage Products

Cleavage products due to the enzymatic activity of the proteolyticenzyme biomarkers of their substrates can also be detected. Cleavageproducts obtained from samples in patients suffering from varying neuralinjuries, degrees of severity of injury, neuronal disorders fragmentsand derivatives thereof, can be prepared as described above.Furthermore, cleavage products can be subjected to enzymatic digestionto obtain fragments or peptides of the biomarkers for the production ofantibodies to different antigenic epitopes that can be present in apeptide versus the whole protein. Antigenic epitopes are useful, forexample, to raise antibodies, including monoclonal antibodies, thatspecifically bind the epitope. Antigenic epitopes can be used as thetarget molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

In a preferred embodiment, the antibodies specifically bind biomarkersidentified by SEQ ID NO's.: 1-148. Epitopes, identified by SEQ ID NO's.:1-148, can be as short as at least about 3 amino acids in length.Antibodies directed to SEQ ID NO's.: 1-148 can also include epitopesthat are directed to the N- and C-terminal regions of biomarkersidentified by SEQ ID NO's.: 1-148. Full length proteins and/or longerfragments than those identified by SEQ ID NO's.: 1-148 are disclosed inTable 1, including accession numbers, for production of antibodies tolonger fragments. The antibodies can be directed to longer N- and/orC-Terminal fragments as identified by SEQ ID NO's.: 1-148 and/orepitopes spanning the identified cleavage sites (see table 2) and can beas short as three amino acids up to 500 amino acids long. See forexample Table 1 for identity of full length proteins. Examples include,but not limited to: achromopeptidase, aminopeptidase, angiotensinConverting Enzyme, bromelain, calpain, calpain I, calpain II,carboxypeptidase A, carboxypeptidase B, carboxypeptidase G,carboxypeptidase P, carboxypeptidase W, carboxypeptidase Y, caspase,caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6,caspase 7, caspase 8, caspase 9, caspase 10, caspase 13, cathepsin B,cathepsin C, cathepsin D, cathepsin G, cathepsin H, cathepsin L,chymopapain, chymase, chymotrypsin, α-clostripain, collagenase,complement C1r, complement C1s, complement Factor D, complement factorI, cucumisin, dipeptidyl peptidase IV, elastase, leukocyte, elastase,pancreatic, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinaseGlu-C, endoproteinase Lys-C, enterokinase, Factor Xa, ficin, furin,granzyme A, granzyme B, HIV Protease, Igase, kallikrein tissue, kinase,leucine aminopeptidases, microsomal, matrix metalloprotease, methionineaminopeptidase, neutrase, papain, pepsin, plasmin, prolidase, pronase E,prostate specific antigen, protease, protease S, proteinase, proteinase3, proteinase A, proteinase K, protein C, pyroglutamate aminopeptidase,thrombin, tissue plasminogen activator, troponins, trypsin, tryptase,urokinase; and, at least one or more peptides identified by SEQ IDNO's.: 1-148.

Cleavage products can be used, for example, to induce antibodiesaccording to methods well known in the art. (See, for instance,Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl.Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Cleaved neural polypeptides comprising one or moreimmunogenic epitopes may be presented for eliciting an antibody responsetogether with a carrier protein, such as an albumin, to an animal system(such as rabbit or mouse), or, if the polypeptide is of sufficientlength (at least about 25 amino acids), the polypeptide may be presentedwithout a carrier. However, immunogenic epitopes comprising as few as 8to 10 amino acids have been shown to be sufficient to raise antibodiescapable of binding to, at the very least, linear epitopes in a denaturedpolypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Nucleic acids cleavage product epitopes can also be recombined with agene of interest as an epitope tag (e.g., the hemaglutinin (“HA”) tag orflag tag) to aid in detection and purification of the expressedpolypeptide. For example, a system described by Janknecht et al. allowsfor the ready purification of non-denatured fusion proteins expressed inhuman cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-897). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the open reading frame of thegene is translationally fused to an amino-terminal tag consisting of sixhistidine residues. The tag serves as a matrix binding domain for thefusion protein. Extracts from cells infected with the recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnand histidine-tagged proteins can be selectively eluted withimidazole-containing buffers.

The antibodies of the present invention may be generated by any suitablemethod known in the art. The antibodies of the present invention cancomprise polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan (Harlow, et al., Antibodies:A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), which is hereby incorporated herein by reference in itsentirety). For example, a polypeptide of the invention can beadministered to various host animals including, but not limited to,rabbits, mice, rats, etc. to induce the production of sera containingpolyclonal antibodies specific for the antigen. The administration ofthe polypeptides of the present invention may entail one or moreinjections of an immunizing agent and, if desired, an adjuvant. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants arealso well known in the art. For the purposes of the invention,“immunizing agent” may be defined as a polypeptide of the invention,including fragments, variants, and/or derivatives thereof, in additionto fusions with heterologous polypeptides and other forms of thepolypeptides as may be described herein.

Typically, the immunizing agent and/or adjuvant will be injected in theanimal by multiple subcutaneous or intraperitoneal injections, thoughthey may also be given intramuscularly, and/or through IV. Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivatizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The antibodies of the present invention can also comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., (1981)), or other methods known to theartisan. Other examples of methods which may be employed for producingmonoclonal antibodies includes, but are not limited to, the human B-cellhybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole etal., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and theEBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies AndCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing the mAb of this invention maybe cultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this the presently preferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a humanimmune system, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The immunizing agent will typically include neural polypeptides,fragments or a fusion protein thereof. Generally, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986), pp.59-103). Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. As inferred throughout the specification,human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc., New York,(1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theneural polypeptides of the present invention. Preferably, the bindingspecificity of monoclonal antibodies produced by the hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay(ELISA). Such techniques are known in the art and within the skill ofthe artisan. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-sepharose, hydroxyapatite chromatography, gel exclusionchromatography, gel electrophoresis, dialysis, or affinitychromatography.

The skilled artisan would acknowledge that a variety of methods exist inthe art for the production of monoclonal antibodies and thus, theinvention is not limited to their sole production in hybridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies, or such chains from human,humanized, or other sources). The hybridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transformed into host cells suchas Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with a biomarker polypeptideor a cell expressing such peptide. Once an immune response is detected,e.g., antibodies specific for the antigen are detected in the mouseserum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention. The antibodies detecting cleavageproducts, peptides and derivatives thereof, can be used in immunoassaysand other methods to identify new cleavage products and for use in thediagnosis of neural injury, degree of severity of injury and/orneurological disorders.

Other methods can also be used for the large scale production ofcleavage product specific antibodies. For example, antibodies can alsobe generated using various phage display methods known in the art. Inphage display methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

The antibodies of the present invention have various utilities. Forexample, such antibodies may be used in diagnostic assays to detect thepresence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay can comprise at least two steps. Thefirst, subjecting a sample with the antibody, wherein the sample is atissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), pp. 147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ²H, ¹⁴C, ³²P, or ¹²⁵I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); David et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Methods, 40:219 (1981); andNygren, J. Histochem. and Cytochem., 30:407 (1982).

Kits

In yet another aspect, the invention provides kits for aiding adiagnosis of neural injury, degree of severity of injury, subcellularlocalization and/or neuronal disorders, wherein the kits can be used todetect the markers of the present invention. For example, the kits canbe used to detect any one or more of the markers described herein, whichmarkers are differentially present in samples of a patient and normalsubjects. The kits of the invention have many applications. For example,the kits can be used to differentiate if a subject has axonal injuryversus, for example, dendritic, or has a negative diagnosis, thus aidingneuronal injury diagnosis. In another example, the kits can be used toidentify compounds that modulate expression of one or more of themarkers in in vitro or in vivo animal models to determine the effects oftreatment.

In one embodiment, a kit comprises (a) a composition or panel ofbiomarkers; (b) a protein substrate; and (c) a detection reagent. Suchkits can be prepared from the materials described above, and theprevious discussion regarding the materials (e.g., antibodies, detectionreagents, immobilized supports, etc.) is fully applicable to thissection and will not be repeated. Optionally, the kit may furthercomprise pre-fractionation spin columns. In some embodiments, the kitmay further comprise instructions for suitable operation parameters inthe form of a label or a separate insert.

In a preferred embodiment, the composition or panel of biomarkersincludes at least one biomarker selected from any one biomarkeridentified by SEQ ID NO's.: 1-148. Preferably, the panel of biomarkersincludes at least two biomarkers selected from SEQ ID NO's.: 1-148 up to145 biomarkers selected from SEQ ID NO's.: 1-148.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Optionally, the kit may further comprise a standard or controlinformation so that the test sample can be compared with the controlinformation standard to determine if the test amount of a markerdetected in a sample is a diagnostic amount consistent with a diagnosisof neural injury, degree of severity of the injury, subcellularlocalization, neuronal disorder and/or effect of treatment on thepatient.

In another embodiment, a kit comprises: (a) a substrate comprising anadsorbent thereon, wherein the adsorbent is suitable for binding amarker, (b) any one biomarker up to one hundred and forty fivebiomarkers selected from biomarkers identified by SEQ ID NO's.: 1-148,and (c) instructions to detect the marker or markers by contacting asample with the adsorbent and detecting the marker or markers retainedby the adsorbent. In some embodiments, the kit may comprise an eluant(as an alternative or in combination with instructions) or instructionsfor making an eluant, wherein the combination of the adsorbent and theeluant allows detection of the markers using gas phase ion spectrometry.Such kits can be prepared from the materials described above, and theprevious discussion of these materials (e.g., probe substrates,adsorbents, washing solutions, etc.) is fully applicable to this sectionand will not be repeated.

In another embodiment, the kit may comprise a first substrate comprisingan adsorbent thereon (e.g., a particle functionalized with an adsorbent)and a second substrate onto which the first substrate can be positionedto form a probe which is removably insertable into a gas phase ionspectrometer. In other embodiments, the kit may comprise a singlesubstrate which is in the form of a removably insertable probe withadsorbents on the substrate. In yet another embodiment, the kit mayfurther comprise a pre-fractionation spin column (e.g., Cibacron blueagarose column, anti-HSA agarose column, size exclusion column, Q-anionexchange spin column, single stranded DNA column, lectin column, etc.).

Optionally, the kit can further comprise instructions for suitableoperational parameters in the form of a label or a separate insert. Forexample, the kit may have standard instructions informing a consumer howto wash the probe after a sample is contacted on the probe. In anotherexample, the kit may have instructions for pre-fractionating a sample toreduce complexity of proteins in the sample. In another example, the kitmay have instructions for automating the fractionation or otherprocesses.

The following examples are offered by way of illustration, not by way oflimitation. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Any one or more of thefeatures of the previously described embodiments can be combined in anymanner with one or more features of any other embodiments in the presentinvention. Furthermore, many variations of the invention will becomeapparent to those skilled in the art upon review of the specification.The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

EXAMPLES Materials and Methods In Vivo Model of Brain Injury Model.

A controlled cortical impact (CCl) device is used to model TBI in ratsand generate brain tissue and CSF samples. Adult male (280-300 g)Sprague-Dawley rats (Harlan: Indianapolis, Ind.) are anesthetized with4% isoflurane in a carrier gas of 1:1 O₂/N₂O (4 min.) followed bymaintenance anesthesia of 2.5% isoflurane in the same carrier gas. Corebody temperature is monitored continuously by a rectal thermistor probeand maintained at 37±1° C. by placing an adjustable temperaturecontrolled heating pad beneath the rats. Animals are mounted in astereotactic frame in a prone position and secured by ear and incisorbars. A midline cranial incision is made, the soft tissues reflected,and a unilateral (ipsilateral to site of impact) craniotomy (7 mmdiameter) is performed adjacent to the central suture, midway betweenbregma and lambda. The dura mater is kept intact over the cortex. Braintrauma is produced by impacting the right cortex (ipsilateral cortex)with a 5 mm diameter aluminum impactor tip (housed in a pneumaticcylinder) at a velocity of 3.5 m/s with a 1.6 mm compression and 150 msdwell time (compression duration). These injuries are associated withdifferent magnitudes of local cortical contusion and more diffuse axonaldamage. Velocity is controlled by adjusting the pressure (compressed N₂)supplied to the pneumatic cylinder. Velocity and dwell time are measuredby a linear velocity displacement transducer (Lucas Shaevitz™ model 500HR; Detroit, Mich.) that produces an analogue signal that is recorded bya storage-trace oscilloscope (BK Precision, model 2522B; Placentia,Calif.). Sham-injured control animals undergo identical surgicalprocedures but do not receive an impact injury. Appropriate pre- andpost-injury management is maintained to insure compliance withguidelines set forth by the University of Florida Institutional AnimalCare and Use Committee and the National Institutes of Health guidelinesdetailed in the Guide for the Care and Use of Laboratory Animals.Different brain tissue regions, CSF, and serum samples are collected at7 a maximum of 7 tie points (6, 12, and 24 hours, and 2, 3, 7, and 14days) after CCI, as described below.

Similarly, multi-organ injury can be induced by sepsis in rats usingmethods well-known in the art.

Brain Tissue Collection and Preparation

At the appropriate time-points (6, 12, 24, 48 and 72 h, 5 and 7 days)after injury, animals are anesthetized and immediately killed bydecapitation. Organ tissue is immediately removed, rinsed with ice coldPBS and halved. Different regions (if desired) are rapidly dissected,rinsed in ice cold PBS, snap-frozen in liquid nitrogen, and frozen at−80° C. until used. For Western blot analysis, the brain samples arepulverized with a small mortar-pastel set over dry ice to a fine powder.The pulverized brain tissue powder is then lysed for 90 min at 4° C.with 50 mM Tris (pH 7.4), 5 mM EDTA, 1% (v/v) Triton X-100, 1 mM DTT, 1×protease inhibitor cocktail (Roche Biochemicals). The brain lysates arethen centrifuged at 8000 g for 5 min at 4° C. to clear and removeinsoluble debris, snap-frozen and stored at −85° C. until used.

SDS-Polyacrylamide Gel Electrophoresis and Electrotransfer.

Protein concentrations of tissue lysates and CSF are determined bybicinchoninic acid microprotein assays (Pierce Inc., Rockford, Ill.,USA) with albumin standards. Protein balanced samples are prepared forsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) intwo-fold loading buffer containing 0.25 M Tris (pH 6.8), 0.2 M DTT, 8%SDS, 0.02% bromophenol blue, and 20% glycerol in distilled H₂O. Twentymicrograms (20 μg) of protein per lane will be routinely resolved bySDS-PAGE on 6.5% Tris/glycine gels for 2 h at 200 V. Followingelectrophoresis, separated proteins will be laterally transferred topolyvinylidene fluoride (PVDF) membranes in a transfer buffer containing0.500 M glycine and 0.025 M Tris-HCl (pH 8.3) 10% methanol at a constantvoltage of 20 V for 2 h at 4° C. in a semi-dry transfer unit (Bio-Rad).

Sandwich ELISA.

Anti-Biomarker specific rabbit polyclonal antibody and monoclonalantibodies are produced in the laboratory. To determine reactivity andspecificity of the antibodies a tissue panel is probed by Western blot.An indirect ELISA is used with the recombinant biomarker proteinattached to the ELISA plate to determine the optimal concentrations ofthe antibodies used in the assay. This assay determines a robustconcentration of anti-biomarker to use in the assay. 96-well microplatewells are coated with 50 ng/well and the rabbit and mouse anti-biomarkerantibodies are diluted serially starting with a 1:250 dilution down to1:10,000 to determine the optimum concentration to use for the assay. Asecondary anti-rabbit (or mouse)-horseradish peroxidase (HRP) labeleddetection antibody and Ultra-TMB are used as detection substrate toevaluate the results.

Once the concentration of antibody for maximum signal are determined,maximum detection limit of the indirect ELISA for each antibody isdetermined. 96-well microplates are coated with a concentration from 50ng/well serially diluted to <1 pg/well. For detection antibodies arediluted to the concentration determined above. This provides asensitivity range for the Biomarker ELISA assays and determines whichantibody to choose for capture and detection antibody.

Optimization and enhancement of signal in the sandwich ELISA: Thedetection antibody is directly labeled with HRP to avoid any crossreactivity and to be able to enhance the signal with the amplificationsystem, which is very sensitive. This format is used in detecting allthe biomarkers. The wells of the 96-well plate are coated withsaturating concentrations of purified antibody (˜250 ng/well), theconcentration of biomarker antigen ranges from 50 ng to <1 pg/well andthe detection antibody is at the concentration determined above.Initially the complex is detected with a HRP-labeled secondary antibodyto confirm the SW ELISA format, and the detection system is replaced bythe HRP-labeled detection antibody.

Standard curves of biomarkers and samples from control and injuredanimals are used. This also determines parallelism between the serumsamples and the standard curve. Serum samples are spiked with a serialdilution of each biomarker, similar to the standard curve. Parallelresults are equal to 80-100% recovery. If any high concentrations ofserum have interfering substances, the minimum dilution required isdetermined to remove the interference. The assay is used to evaluatebiomarker levels in serum from injured animals having injuries ofdifferent magnitudes followed over time.

Immunoblotting Analysis

After electrotransfer, blotting membranes are blocked for 1 h at ambienttemperature in 5% non-fat milk in TBS and 0.05% Tween-2 (TBST), thenincubated in primary monoclonal or polyclonal antibody in TBST with 5%milk (see list below, at 1/1000 to 1/3,000 dilution as recommended bythe manufacturer) at 4° C. overnight, followed by four washes with TBSTand a 2 hour incubation at ambient temperature with either a secondaryantibody linked to horseradish peroxidase (enhanced chemiluminescence,(ELC) method) or biotinylated secondary antibody (Amersham) followed bya 30 min incubation with strepavidin-conjugated alkaline phosphatase(colorimetric method). ECL reagents (Amersham) are used to visualize theimmunolabeling on x-ray film. Alternatively, colorimetric development isperformed with a one-step BCIP-reagent (Sigma). Molecular weight ofintact proteins and their potential breakdown products (BDP) areassessed by running along side rainbow colored molecular weightstandards (Amersham). Semi-quantitative evaluation of protein and BDP'slevels are performed via computer-assisted densitometric scanning (EpsonXL3500 high resolution flatbed scanner) and image analysis with Image Jsoftware (NIH).

In vivo cleavage sites identification: To identify the major in vivocleavage sites of each proteolysis-prone axonal and myelin structuralprotein, brain tissue is pooled from injured rats at a time point thatoptimally produced the major BDP. To obtain the maximal yield, tissuesampling combines ipsilateral cortex, subcortical white matter andCorpus callosum. The pooled brain lysate using Triton X-100 is preparedas described under: Brain tissue collection and preparation (see above).The lysate (5 mg protein) is subjected to strong anion exchangechromatography (1.0 mm diameter, 10 cm long) using the BioRad DualflowBiological system. The bound proteins are eluted with a NaCl gradient20-500 mM) and 30-50 fractions collected. Fractions containing thespecific proteins and fragments of interest are determined by subjecting20 microliter of each fractions to SDS-PAGE Western blot analysis. Inthis manner, both the intact protein substrate and its major BDP(s) willbe isolated. These proteins are then further separated by SDS-PAGE andelectrotransfer to PVDF members. The now separated BDP and intactprotein bands are visualized Coomassie blue staining (80% Methanol, 5%acetic acid and 0.05% Coomassie brilliant blue R250 for 1 min. The BDPband is cut out and subjected to N-terminal microsequencing ortrypsinization and mass spectrometry analysis to identify it newN-terminal. By matching the sequence generated from proteomic analysiswith the full-length protein sequences in the rat proteome database withbioinformatic tools such as MASCOT, the major cleavage site of theprotein substrate can be identified. Using this method, cleavage sitesfrom proteins of interest have been identified (see Table 1 and Table2).

In Vitro Protease Digestion and Cleavage Site Identification

In vitro protease digestion of purified tissue proteins (10-50 μg) withpurified proteases (0.1-2.5 μg) (human calpain-1 and porcine calpain-2(Calbiochem), recombinant human caspase-3 caspase-6 and 7 (Chemicon),bovine cathepsin B (Sigma) and human MMP-2 and -9 (Chemicon) wasperformed with a substrate to protease ratio of 1/200 to 1/40 for 2hours to overnight in a buffer containing 50 mM HEPES (pH 7.4) with theexception of cathepsin B (with which we use 100 mM MES, pH 5.5). 10 mMdithiothreitol was also added for cysteine proteases (calpains, caspasesand cathepsin B) but not MMP's. In addition, 1 mM EDTA was added forcaspases while 2 mM excess CaCl₂ is added. The protease reaction isstopped by the addition of a protease inhibitor cocktail (Roche). Thedigested brain lysate is subjected to immunoprecipitation and cleavagesite identification as described above. The in vitro cleavage pattern ofa specific axonal or myelin protein of interest are compared side-byside with the in vivo TBI-induced cleavage pattern by Western blotanalysis (10 μg).

Preparation of Novel Fragment-Specific Antibodies and ELISA Developmentand Usage (a) Generation of Fragment-Specific Polyclonal Antibodies

Once the new N-terminal of a BDP can be identified, it is possible toraise fragment-specific antibody using our established and uniquetechnique.

TBDP-26 kDa ARVAGVS₁₂₁{circumflex over ( )}K₁₂₂DRTGN ARVAGVS-_(COOH);SEQ ID NO: 146 SEQ ID NO: 145 _(NH2)-KDRTGNDE SEQ ID NO: 147Using tau protein as an example, tau has an unique cleavage site(ARVAGVS₁₂₁̂K₁₂₂DRTGN) (SEQ ID NO: 145), synthetic peptideNH2-Cys-C₆₋ARVAGVS_(-COOH) (SEQ ID NO: 146) and(_(NH2)-KDRTGNDE-C₆-Cys-OH) (SEQ ID NO: 147) based on the new C-terminalor N-terminal of the two Tau-BDPs-26 kDa are custom-made (CaliforniaPeptide, Napa, Calif.). A C₆ linker and a C-terminal cysteine [C] willbe introduced for the subsequent coupling of the peptide to KeyholeLimpet hemocyanin (KLH) protein using a sulfo-link crosslinking reagent(Pierce). Following coupling efficiency determinations, peptides aredialyzed, concentrated and 2 mg of conjugated protein will be used formultiple antigen injections into 2 rabbits. After 3 months, serumsamples from the rabbits are collected and subjected to affinitypurification using NH₂-Cys-C₆₋ARVAGVS_(-COOH) or(_(NH2-)KDRTGNDE-C₆-Cys-OH) coupled to sulfo-linked resins (Pierce). Theaffinity-purified antibody is dialyzed against TBS (20 mM Tris-HCl, pH7.4, 150 mM NaCl), concentrated and stored in 50% glycerol at −20° C.Confirmation of the specificity of antibodies for Tau-BDPs-26 kDaemploys Western blot comparisons to BDPs assessed with a monoclonalantibody to total αII-spectrin following challenge of rats with mild andmoderate TBI, as well as cell or brain lysate digested by differentproteases (such as calpain or caspases), a technique we previouslyemployed. Similarly, monoclonal antibodies can also be generated.

(b) Fragment-Specific ELISA's Development and Usage

To illustrate the method of making fragment-specific enzyme-linkedimmunoassay (ELISA), Tau-BDP-26 kDa is used as an example: First Westernblotting is used to confirm the immunoreactivity of commercialmonoclonal antibodies that recognize rat tau and its BDPs (Cedarlane,clone Tau-1) and polyclonal anti-Tau-BDP-26 kDa antibodies. 10 μg/ml ofthe polyclonal anti-Tau-BDP-26 kDa antibody is used as capture antibodyto coat 96-well ELISA microplate wells in 0.1M carbonate-bicarbonateBuffer pH 9.8 overnight. After rinsing each well with 200 μL PBS with0.05% Tween 20 three times, non-specific sites are blocked with blockingbuffer (0.1% BSA, 0.05% Tween 20 in PBS) for 1 hour at room temperature.Brain tissue lysate (1-25 μg) or CSF samples (2.5-10 μl) from controland TBI animals are diluted with blocking buffer to 100 μl and added toeach well. The plate is incubated on shaker at 150 rmp, 26° C. for 30minutes to 2 h. After rinsing each well with 200 μl PBS with 0.05% Tween20 three times, the wells are probed with detecting commercialmonoclonal antibodies that recognize rat tau and its BDPsfragment-specific antibody at 1 μg/ml with blocking buffer (100 μl) andincubated at 150 rpm, 26° C. for 30-60 minutes. The washing steps arerepeated and the plate probed with HRP-coupled secondary antibody(1/20,000) (Pierce), washed again before developing with 100 μl of TMBsubstrate solution (Ultra-sensitive ABC peroxidase Reagent, Pierce)(Pierce prod# 34028) with incubation at 150 rpm, 26° C. for 5-30minutes. After the addition of 50-μl stop solution (1N H₂SO₄) to eachwell (5 min), the plate is read at OD₄₅₀ nm with a spectrophotometer.This sandwich ELISA format selectively detects Tau-BDP-26 kDa but notthe intact Tau or other BDP's present in biological samples.

Example 1 Detection of Neural Proteins Subjected to Proteolytic Attack48 h after Traumatic Brain Injury (TBI) in Rats

TBI was induced in rodents as described above. 48 h following TBI orsham operation or naïve rats, samples of CSF were collected and analyzedfor presence of five novel neural protein markers (NF-H (A), NF-L (B),Tau (C), APP (D) and βII-spectrin (E) (FIG. 2) were identified to bevulnerable to endogenous proteolytic attack, producing major breakdownproducts (BDPs) in the ipsilateral hippocampus. Ipsilateral corticalsamples were also analyzed and they showed very similar patterns ofproteolysis. These unique BDP's when accumulated in biofluids such asCSF and blood, can be readily detected by immunological techniques suchas Western blots or ELISA and thus are excellent diagnostic markers fororgan-specific (brain or spinal cord or peripheral nerve) injury orstress (FIG. 1).

Example 2 Detection of Myelin Proteins Subjected to Proteolytic Attack48 h after Traumatic Brain Injury (TBI) in Rats

TBI was induced in rodents as described above. 48 h following TBI orsham operation or naïve rats, samples of CSF were collected and analyzedfor presence of two novel myelin sheath protein markers (MBP and MOSP)(FIG. 3) were identified to be vulnerable to endogenous proteolyticattack, producing major breakdown products (BDPs) in the ipsilateralhippocampus. Ipsilateral cortical samples were also analyzed and theyshowed very similar patterns of proteolysis. Based on the uniquecleavage site in MBP (DENPVVHFF₁₁₄̂K₁₁₅NIVTPP) (SEQ ID NO: 148), we haveproduced polyclonal and monoclonal that specifically detects the newN-terminal (NH2-KNIVTPP) (SEQ ID NO: 149) of MBP-BDP of 12 kDa and 10kDa (FIG. 4). These unique BDPs when accumulated in biofluids such asCSF and blood are excellent diagnostic markers for organ-specific(brain, spinal cord or peripheral nerve) injury or stress and can bedetected by techniques using fragment-specific antibody tools such asthose described in FIG. 4 by Western blots or ELISA.

Example 3 Detection of Synaptic Proteins (Synaptotagmin andSynaptojanin-1) are Being Degraded in Rat Cortex and/or Hippocampus 48hr after TBI in Rats

TBI was induced in rodents as described above. 48 h following TBI orsham operation or naïve rats, samples of CSF were collected and analyzedfor presence of five novel synaptic protein markers (Synaptotagmin and)(top), and Synaptojanin-1 (bottom) (FIG. 5) were identified to bevulnerable to endogenous proteolytic attack, producing major breakdownproducts (BDPs) in the ipsilateral hippocampus. Ipsilateral corticalsamples were also analyzed and they showed very similar patterns ofproteolysis. These unique BDP's when accumulated in biofluids such asCSF and blood, detected by immunological techniques such as Westernblots or ELISA and thus are excellent diagnostic marker fororgan-specific (brain or spinal cord or peripheral nerve) injury orstress (FIG. 1).

Example 4 Detection of Major Cardiac Troponin-T2 and Troponin-I32 wereCleaved by Calpain-2 and Caspase-3 Proteases, Producing Unique BreakdownProducts

Cardiac stress, heart failure or cardiac ischemia induce overactivationof proteases such as calpain and caspase-3. Here we used purified humancardiac isoforms of troponinT2 and troponin-I3 are subjected them to invitro incubation with calpain and/or caspase-3. We found that bothtroponins are vulnerable to proteolytic attack (FIG. 6) producing majorbreakdown products (BDPs) in the process. We expect that the same BDP'swill be produced in cardiac injury or stressing animals and in humans.These unique BDP's when accumulated in biofluids such as blood, can bereadily detected by immunological techniques such as Western blots orELISA and thus are excellent diagnostic markers for organ-specific(heart) injury or stress (FIG. 1).

Example 5 Detection of Unobvious and Unique Cleavage Sites of MajorOrgan or Tissue Proteins in Stressed or Injured Animals or During InVitro Protease Incubation

Using the methods described above and similar to outlined in example1-3, at least 43 unobvious and unique cleavage sites of major organ ortissue proteins (Table 2; SEQ ID NO's.: 1-148) have been identified. Theexact cleavage sites enable the synthesis of peptides that mimics thenew C-terminal or new N-terminal epitope and these can then be used togenerate fragment-specific antibodies or other capture or detectingagents. These unique BDP's when accumulated in biofluids such as blood,can be readily detected by the methods described herein, e.g.immunological techniques such as Western blots or ELISA and thus areexcellent diagnostic marker for organ-specific (heart) injury or stress(FIG. 1).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

TABLE 2 Examples of unobvious and unique tissue protein cleavage sitesproduced by protease attack Molec- Molec- Example of new ular ular C-andN-terminal weight Spe- weight epitopes fragment- Protein Protein Acc. #cies BDP BDP Cleavage site identified specific antibodies alphaII- 260kDa hypothe- Dog SBDP150- 150 kDa WRSLQQLAEERSEVY₁₁₇₃*G₁₁₇₄WRSLQQLAEERSEVY _(-COOH); spectrin tical short MMPRDDTDSKTASP SEQ ID NO:49 SEQ ID No.: 1 _(NH2-)GMMPRDDTDSKTASP SEQ ID NO: 50 Fast  33 kDaNP_006748 Human TnT3BDP  22 kDa AEEDAEEEKPRPKLT₄₄*A₄₅PKIAEEDAEEEKPRPKLT_(COOH); skeletal PEGEKVDFDDIQ SEQ ID NO: 51 muscle SEQID NO: 2 _(NH2)APKIPEGEKVDFDDIQ Troponin- SEQ ID NO: 52 T3 Fast  22 kDaNP_003273 Human TnI2BDP <10 kDa PPLRRVRMSADAMLK₁₂₄*A₁₂₅LPPLRRVRMSADAMLK_(COOH) sketal LGSKHKVCMDLRAN SEQ ID NO: 53 muscle SEQ IDNO: 3 _(NH2)ALLGSKHKVCMDLRAN Troponin SEQ ID NO: 54 I-2 (1) Fast  22 kDaNP_003273 Human TnI2BDP <10 kDa GSKHKVCMDLRANLK₁₄₂*Q₁₄₃VGSKHKVCMDLRANLK_(COOH) sketal KKEDTEKERDLRDV SEQ ID NO: 55 muscle SEQ IDNO: 4 _(NH2)QVKKEDTEKERDLRDV Troponin SEQ ID NO: 56 I-2 (2) Fast  22 kDaNP_003273 Human TnI2BDP <10 kDa EEEKYDMEVRVQKTS₉₀*K₉₁ELEEEEKYDMEVRVQKTS_(COOH) sketal DMNQKLFDLRGK SEQ ID NO: 57 muscle SEQ IDNO: 5 _(NH2)KELEDMNQKLFDLRGK Troponin SEQ ID NO: 58 I-2 (3) Slow  33 kDaNP_003274 Human TnT1BDP  12 kDa VLSNMGAHFGGYLVK₁₆₇*A₁₆₈VLSNMGAHFGGYLVK_(COOH) skeletal EQKRGKRQTGREMKV SEQ ID NO: 59 muscle SEQID NO: 6 _(NH2)AEQKRGKRQTGREMKV Troponin- SEQ ID NO: 60 T1 Slow/Fast  22kDa NP_006748 Human TnT3BDP <10 kDa TREIKDLKLKVMDLR₁₁₅*G₁₁₆TREIKDLKLKVMDLR_(COOH) skeletal KFKRPPLRRVRVSA SEQ ID NO: 61 muscle SEQID NO: 7 _(NH2)GKFKRPPLRRVRVSA Tropoinin SEQ ID NO: 62 I1 and I2 Fast 22 kDa NP_003273 Human TrI1BDP KELEDMNQKLFDLRG₁₀₆↓K₁₀₇KELEDMNQKLFDLLRG_(-COOH); skeletal FKRPPLRRVRMSAD SEQ ID NO: 63 muscleSEQ ID NO: 8 _(NH2-)KFKRPPLRRVRMSAD Troponin- SEQ ID NO: 64 I1 Fast  33kDa NP_006748 Human TnT3BDP PKLTAPKIPEGEKVD₅₅↓F₅₆PKLTAPKIPEGEKVD_(-COOH); skeletal DDIQKKRQNKDLME SEQ ID NO: 65 muscleSEQ ID NO: 9 _(NH2-)FDDIQKKRQNKDLME Troponin- SEQ ID NO: 66 T3 MAP-2A/B/300 kDa CAA37535 Rat 150, ADRETAEEVSARIVQ₉₆*V₉₇ ADRETAEEVSARIVQ_(-COOH);C/D  90 kDa VTA EAVAVLKGEQE SEQ ID NO: 67 SEQ ID NO: 10_(NH2-)VVTAEAVAVLKGEQE SEQ ID NO: 68 MBP  18-21 CAA10805 Rat M-BDP-KSQRTQDENPVVHFF₁₁₄{circumflex over ( )}K₁₁₅ KSQRTQDENPVVHFF-COOH; kDa*10-12 NIVTPPRTPPPSQG SEQ ID NO: 69 kDa SEQ ID NO: 11_(NH2-)K115NIVTPPRTPPPSQG- COOH SEQ ID NO: 70 NF-L  68 kDa AAH39237Human NFL-BDP  30 kDa KSRFTVLTESAAKNTD₂₉₇{circumflex over ( )}A₂₉₈KSRFTVTESAAKNTD_(COOH); VRAAKDEVSESRRL SEQ ID NO: 71 SEQ ID NO: 12_(NH2-)AVRAAKDEVSESSRL SEQ ID NO: 72 NF-L  68 kDa AAH39237 Human NFL-BDP 31 kDa NAEEWFKSRFTVLTE₂₉₀*S₂₉₁ NAEEWFKSRFTVLTE_(-COOH); AAKNTDAVRAAKDESEQ ID NO: 73 SEQ ID NO: 13 _(NH2-)SAAKNTDAVRAAKDE SEQ ID NO: 74 NF-M150 kDa NP_005373 Human NFM-BDP  80 kDa QAEEWFKCRYAKLTE₃₀₀{circumflexover ( )}A₃₀₁ QAEEWFKCRYAKLTE_(-COOH); AEQNKEAIRSAKEE SEQ ID NO: 75 SEQID NO: 14 _(NH2-)A AEQNKEAIRSAKEE; SEQ ID NO: 76 NF-M 150 kDa NP_005373Human NFL-BDP-  32 kDa ALKEIRSQLESHSDQ₂₈₃*N₂₈₄ ALKEIRSQLESHSDQ _(--COOH); MHQAEEWFKCRYAK SEQ ID NO: 77 SEQ ID NO: 15 _(NH2-) NMHQAEEWFKCRYAK SEQID NO: 78 Prion  28 kDa AAX42952 human PrBDP AAGALVGGLGGYMLG₁₃₁*S₁₃₂AAGALVGGLGGYMLG_(-COOH) Protein AMSRPIIHFGSDYE SEQ ID NO: 79 SEQ ID NO:16 _(NH2)SAMSRPIIHFGSDYE SEQ ID NO: 80 Slow  22 kDa NP_003273 HumanTrI2BDP REIKDLKLKVMDLRG₁₀₇↓K₁₀₈ REIKDLKLKVMDLRG_(-COOH); skeletalFKRPPLRRVRVSAD SEQ ID NO: 81 muscle SEQ ID NO: 17 _(NH2-)KFKRPPLRRVRVSADTroponin- SEQ ID NO: 82 I2 Slow 33 kDa NP_003274 Human TnT1BDPPPLIPPKIPEGERVD₅₀↓F₅₁ PPLIPPKIPEGERVD_(-COOH); skeletal DDIHRKRMEKDLLESEQ ID NO: 83 muscle SEQ ID NO: 18 _(NH2-)FDDIHRKRMEKDLLE Troponin- SEQID NO: 84 T1 Tau 441-  50 kDa NP_058908 Rat TBDP  26 kDaQAAGHVTQARVAGVS₁₂₁{circumflex over ( )}K₁₂₂ QAAGHVTQARVAGVS_(-COOH);isoform DRTGNDEKKAKGADG SEQ ID NO: 85 SEQ ID NO: 19_(NH2-)KDRTGNDEKKAKGADG SEQ ID NO: 86 Tau 441-  50 kDa AAC04279 HumanTBP  23 kDa EDEAAGHVTQARMVS₁₃₀*K₁₃₁ EDEAAGHVTQARMVS_(-COOH); isoformSKDGTGSDDKKAKG SEQ ID NO: 87 SEQ ID NO: 20 _(NH2-)KSKDGTGSDDKKAKG SEQ IDNO: 88 Tau 441-  50 kDa AAC04279 Human TBP AKGADGKTKIATPRG₁₅₇*A₁₅₈AKGADGKTKIATPRG_(-COOH); isoform APPGQKGQANATRIP SEQ ID NO: 89 SEQ IDNO: 21 _(NH2-)APPGKQGQANATRIP SEQ ID NO: 90 Tau 441-  50 kDa AAC04279Human TBP GGGNKKIETHKLTFR₃₈₀*E₃₈₁ GGGNKKIETHKLTFR_(-COOH); isoformNAKAKTDHGAEIVH SEQ ID NO: 91 SEQ ID NO: 22 _(NH2-)ENAKAKTDHGAEIVH SEQ IDNO: 92 Activated  80 kDa AAV41878 Human BDP  76 kDaGVSAQVQKQRARELG₂₇*L₂₈ GVSAQVQKQRARELG_(-COOH); Calpain-1 GRHENAIKYLGQDYSEQ ID NO: 93 SEQ ID NO: 23 _(NH2-)LGRHENAIKYLGQDY SEQ ID NO: 94Activated  80 kDa CIHUH2 Human BDP  41 kDa RGSTAGGCRNYPNTF₃₈₁*W₃₈₂RGSTAGGCRNYPNTF_(-COOH); Calpain-2 MNPQYLIKLEEEEE SEQ ID NO: 95 SEQ IDNO: 24 _(NH2-)WMNPQYLIKLEEEEE SEQ ID NO: 96 alphaII- 260 kDa Q13813Mouse SBDP150- 150 kDa LMAEEVQAVQQQEVY₁₁₇₆*G₁₁₇₇ LMAEEVQAVQQQEVY_(-COOH); spectrin short AMPRDETDSKTASP SEQ ID NO: 97 SEQ ID NO: 25_(NH2-)GAMPR DETDSKTASP SEQ ID NO: 98 alphaII- 260 kDa Q13813 HumanSBDP150- 150 kDa LMAEEVQAVQQQEVY₁₁₇₆*G₁₁₇₇ LMAEEVQAVQQQEVY _(-COOH);spectrin short MMPRDETDSKTASP SEQ ID NO: 99 SEQ ID NO: 26_(NH2-)GMMPRDETDSKTASP SEQ ID NO: 100 alphaII- 260 kDa Q13813 HumanSBDP145 145 kDa RSLQQLAEERSQLLG₁₂₃₀*S₁₂₃₁ RSLQQLAEERSQLLG _(-COOH);spectrin (Cal- AHEVQRFHRDADET SEQ ID NO: 101 pain) SEQ ID NO: 27_(NH2-)SAHEVQRFHRDADET SEQ ID NO: 102 alphaII- 260 kDa Q13813 HumanSBDP149 149 kDa QQQEVYGMMPRDETD₁₁₈₅*S₁₁₈₆ QQQEVYGMMPRDETD _(-COOH);spectrin (Cas- KTASPWKSARLMVH SEQ ID NO: 103 pase) SEQ ID NO: 28_(NH2-)SKTASPWKSARLMVH SEQ ID NO: 104 alphaII- 260 kDa Q13813 HumanSBDP120 120 kDa REAFLNTEDKGDSLD₁₄₇₈*S₁₄₇₉ REAFLNTEDKGDSLD _(-COOH);spectrin (Cas- VEALIKKHEDFDKA SEQ ID NO: 105 pase) SEQ ID NO: 29_(NH2-)SVEALIKKHEDFDKA SEQ ID NO: 106 BAX  21 kDa NP_620119 Human  18kDa SSEQIMKTGALLLQG₂₉↓F₃₀ SSEQIMKTGALLLQG_(-COOH); IQDRAGRMGGEAPE SEQ IDNO: 107 SEQ ID NO: 30 _(NH2-)FIQDRAGRMGGEAPE SEQ ID NO: 108 betaII- 240kDa* NP_003119 Human βIISBDP 110 kDa ENQMEVRKKEIEELQ₁₄₄₀S₁₄₄₁ENQMEVRKKEIEELQ_(-COOH); spectrin QAQALSQEGKSTED SEQ ID NO: 109 SEQ IDNO: 31 _(NH2-)SQAQALSQEGKSTED SEQ ID NO: 110 betaII- 240 kDa* NP_003119Human βIISBDP 108 kDa AQALSQEGKSTDEVD₁₄₅₇*S₁₄₅₈ AQALSQEGKSTDEVD_(-COOH);spectrin KRLTVQTKFMELLE SEQ ID NO: 111 SEQ ID NO: 32_(NH2-)SKRLTVQTKFMELLE SEQ ID NO: 112 betaII- 240 kDa* NP_003119 HumanβIISBDP  24 kDa LPAEQGSPRMAETVD₂₁₄₆*T₂₁₄₇ LPAEQGSPRMAETVD_(-COOH);spectrin SEMVNGATEQRTSS SEQ ID NO: 113 SEQ ID NO: 33_(NH2-)T2147SEMVNGATEQRTSS SEQ ID NO: 114 betaII- 240 kDa* NP_003119Human βIISBDP 109 kDa KKEIEELQSQAQALS₁₄₄₈*Q₁₄₄₉ KKEIEELQSQAQALS_(-COOH);spectrin EGKSTDEVDSKRLT SEQ ID NO: 115 SEQ ID NO: 34_(NH2-)QEGKSTDEVDSKRLT SEQ ID NO: 116 betaII- 240 kDa* NP_003119 HumanβIISBDP 107 kDa TDEVDSKRLTVQTKF₁₄₆₇*M₁₄₆₈ TDEVDSKRLTVQTKF_(-COOH);spectrin ELLEPLNERKHNLL SEQ ID NO: 117 SEQ ID NO: 35 _(NH2-)MEL LEPLNERKHNLL SEQ ID NO: 118 betaII- 240 kDa* NP_003119 Human βIISBDP 105 kDaMELLEPLNERKHNLL₁₄₈₂*A₁₄₈₃ MELLEPLNERKHNLL_(-COOH); spectrinSKEIHQFNRDVEDE SEQ ID NO: 119 SEQ ID NO: 36 _(NH2-)ASKEIHQFNRDVEDE SEQID NO: 120 Calcineurin  61 kDa Q08209 Human BDP  45 kDaICSDDELGSEEDGFD₃₈₅*G₃₈₆ ICSDDELGSEEDGFD_(-COOH); A alpha) ATAAARKEVIRNKISEQ ID NO: 121 SEQ ID NO: 37 _(NH2-)GATAAARKEVIRNKI SEQ ID NO: 122CaMPK-IV  55 kDa NP_033923 Mouse BDP  33 kDa VTASTENLVPDYWID₃₀*G₃₁VTASTENLVPDYWID_(-COOH); SNRDPLGDFFEVES SEQ ID NO: 123 SEQ ID NO: 38_(NH2-)GSNRDPLGDFFEVES SEQ ID NO: 124 CaMPK-IV  55 kDa NP_033923 MouseBDP  34 kDa CPSSPCSSVTASTEN₂₃*L₂₄ CPSSPCSSVTASTEN_(-COOH);VPDYWIDGSNRDPL SEQ ID NO: 125 SEQ ID NO: 39 _(NH2-)LVPDYWIDGSNRDPL SEQID NO: 126 CaMPK-IV  55 kDa NP_033923 Mouse BDP  38 kDaKPELLYATPAPDAP₁₇₆*L₁₇₇ KEPNLLYATPAPDAP_(-COOH); KIADFGLSKIVEHQ SEQ IDNO: 127 SEQ ID NO: 40 _(NH2-)LKIADFGLSKIVEHQ SEQ ID NO: 128 CaMPK-IV  55kDa NP_033923 Mouse BDP  40 kDa SKIVEHQVLMKTVCG₂₀₁*T₂₀₂SKIVEHQVLMKTVCH_(-COOH); PGYCAPEILRGCAY SEQ ID NO: 129 SEQ ID NO: 41_(NH2-)TPGYCAPEILRGCAY SEQ ID NO: 130 Cardiac  24 kDa P19429 Human BDP 10 kDa TEIADLTQKIFDLRG₁₃₈↓K₁₃₉ TEAIDLTQKIFDLRG_(-COOH); Troponin-FKRPTLRRVRISAD SEQ ID NO: 131 I3 SEQ ID NO: 42 _(NH2-)KFKRPTLRRVRISADSEQ ID NO: 132 Cardiac  35 kDa NP_00355 Human BDP  24 kDaMEESKPKPRSFMPNL₈₅↓V₈₆ MEESKPKPRSFMPNL_(-COOH); Troponin- PPKIPDGERVDFDDSEQ ID NO: 133 T2 SEQ ID NO: 43 _(NH2-)V86PPKIPDGERVDFDD SEQ ID NO: 134Cardiac  35 kDa NP_00355 Human BDP  13 kDa PNLVPPKIPDGERVD₉₇↓F₁₉₈PNLVPPKIPDGERVD_(-COOH); Troponin- DDIHRKRMEKDLNE SEQ ID NO: 135 T2 SEQID NO: 44 _(NH2-)F98DDIHRKRMEKDLNE SEQ ID NO: 136 NF-M 145 kDa AY421963Bo- NF-M BDP 112 KVEDEK₄₆₇*S₄₆₈SEMEEAL KVEDEK₄₆₇* vine SEQ ID NO: 45 SEQID NO: 137 S₄₆₈SEMEEAL SEQ ID NO: 138 NF-M 145 kDa AY421963 Bo- NF-M BDP110 KKSPVK₅₁₆*A₅₁₇TAPELK KKSPVK₅₁₆ vine SEQ ID NO: 46 SEQ ID NO: 139A₅₁₇TAPELK SEQ ID NO: 140 Tau 441-  50 kDa AAC04279 Human TBP  38 kDaHLSNVSSTGSIDMVD₃₃₃*S₃₃₄ HLSNVSSTGSIDMVD_(-COOH); isoform PQLATLADEVSASLSEQ ID NO: 141 SEQ ID NO: 47 _(NH2-)SPQLATLADEVSASL SEQ ID NO: 142 Tau441-  50 kDa AAC04279 Human TBP  40 kDa MEDHAGTYGLGDRKD₂₆*Q₂₇MEDHAGTYGLGDRKD_(-COOH); isoform GGYTMHQDGEGDTD SEQ ID NO: 143 SEQ IDNO: 48 _(NH2-)QGGYTMHQDGEGDTD SEQ ID NO: 144

1. A composition of biomarkers comprising at least one prion proteinselected from the group consisting of intact prion protein (Prp), prionprotein isoforms (Prp(C) and Prp(Sc), prion protein family members(PrPLP/DOPPEL, and Shadoo) and calpain/caspase proteolytic breakdownproducts of prion protein or prion protein family members in a suitableexcipient.
 2. A composition of biomarkers comprising one or moretroponin proteins, troponin isoforms or calpain/caspase proteolyticbreakdown products of troponin.
 3. The composition of claim 2, whereinthe troponin proteins are troponin-T, troponin-T or troponin-C.
 4. Thecomposition of claim 3, wherein the troponin proteins are selected fromthe group consisting of troponin-T1 (slow; TNNT1), troponin-T3 (fast;TNNT3), troponin-T2 (cardiac; TNNT2), troponin-I1 (slow; TNNI1),troponin-I2 (fast; TNNI2), troponin-I3 (cardiac; TNNI3), troponin C(TN-C; slow and cardiac) and troponin C(STNC; fast).
 5. The compositionof claim 1, wherein the proteolytic breakdown products of prion proteincomprise one or both polypeptides having the sequence of SEQ ID NO: 79and SEQ ID NO:80.
 6. An antibody composition comprising a suitableexcipient and an antibody that specifically binds either to apolypeptide having the sequence of SEQ ID NO: 79 or to a polypeptidehaving the sequence of SEQ ID NO:
 80. 7. An antibody compositioncomprising a suitable excipient and an antibody that specifically bindsto a polypeptide having the sequence of SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ IDNO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 132, SEQID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 or to apolypeptide having the sequence of SEQ ID NO:
 137. 8. A kit fordiagnosing neural injury and/or neuronal disorders in a subject, the kitcomprising: (a) a substrate for holding a biological sample isolatedfrom a human subject suspected of having an abnormal neural condition,(b) an agent that specifically binds at least one or more of a prionprotein, prion protein calpain breakdown products, troponin protein ortroponin protein calpain/caspase breakdown products; and (c) printedinstructions for reacting the agent with the biological sample or aportion of the biological sample to detect the presence or amount of aprion or troponin protein in the biological sample, wherein the presenceof said prion or troponin protein in amounts greater than in arespective sample from a normal subject is diagnostic of a neuralinjury.
 9. The kit of claim 8, wherein the biological sample is a tissueor biological fluid sample.
 10. The kit of claim 9, wherein thebiological fluid sample is blood or CSF.
 11. The kit of claim 10,wherein the blood sample is serum or plasma.
 12. The kit of claim 8,wherein the substrate is a container, or a hydrophobic, hydrophilic,charged, polar, or metal ion matrix.
 13. The kit of claim 8, wherein theagent is an antibody, single or double stranded oligonucleotide, aminoacid, protein, or peptide.
 14. The kit of claim 8, wherein the one ormore prion or troponin proteins are detected using an immunoassay. 15.The kit of claim 14, wherein the immunoassay is an ELISA.
 16. The kit ofclaim 15, wherein the ELISA is a sandwich assay.
 17. The kit of claim 8,wherein the prion protein is selected from intact prion protein, prionprotein isoforms or calpain proteolytic breakdown products of prionprotein.
 18. The kit of claim 8, wherein the prion protein breakdownproducts are one or more of a polypeptide having the sequence of SEQ IDNO: 79 or SEQ ID NO:
 80. 19. The kit of claim 8, wherein the troponinbreakdown products are one or more of a polypeptide having SEQ ID NO:132, SEQ ID NO: 133 SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 orSEQ ID NO:
 137. 20. The kit of claim 8, wherein the antibody agentindependently and specifically binds to the at least one prion ortroponin protein.
 21. The kit of claim 8, wherein the antibody agentindependently and specifically binds intact prion protein, prion proteinisoforms, proteolytic breakdown products of prion protein, intacttroponin protein, troponin protein isoforms or proteolytic breakdownproducts of troponin protein.
 22. The kit of claim 8, wherein theabnormal neural condition is Alzheimer's Disease, Parkinson Disease,dementia, cardiac stress, heart failure or cardiac ischemia.